R2R1/2 In Diagnosis and Therapy

ABSTRACT

The present invention stems from the finding that two genes designated R2R 1  and R2R 2 , play important roles in tissue development and cancer biology. In particular, the inventors have discovered that these two genes are expressed in pulmonary cells and are required for late branching morphogenesis of pulmonary epithelium and endothelium and support the development/maintenance of the refined three dimensional architecture of the lung. These genes are essential in the squamous differentiation program and development/maintenance of the progenitor (Krt14 expressing) cell pool. Moreover, the inventors have identified crucial roles for these genes in cancer biology, particularly processes associated with the acquisition of an immortal and metastatic phenotype (including cancer progression and metastasis) and pulmonary and cardiac development. Accordingly, the invention provides compounds and methods for use in the treatment of cardiac and pulmonary diseases and well as in cancer.

FIELD OF THE INVENTION

The present invention provides novel genes that support thedevelopment/maintenance of the refined three-dimensional architecture ofthe lung as well as medicaments and compositions for treating pulmonarydiseases and/or conditions.

BACKGROUND OF THE INVENTION

The expression of intermediate filament (Krt6), EDC (EpidermalDifferentiation Complex) and SCC (Stratified epithelium-secreted proteingene complex) genes permits cellular survival in hostile environments.This expression profile leads to squamous differentiation and is ahallmark of skin epithelial cells. The inventors found that thistranscriptional program is also vital for the refinement of pulmonaryarchitecture (late branching morphogenesis): cells in distal airways andblood vessels need to assume a flat shape and acquire mechanicalflexibility in an oxygen-rich environment. Proteins encoded by theintermediate filament group and associated EDC and SCC genes permit thiskind of cell shape and flexibility. At the same time, the lung needs tomaintain progenitor cells that are able to differentiate in cellscapable of building such proteins. These progenitor or basal cellstypically express the intermediate filament gene Krt14.

The lung is exposed to vast amounts of mechanical and oxidative stressand resembles the skin in this respect. The squamous differentiationprogram as mentioned above is the primary line of defence. Cells thatline the distal airways and blood vessels of the lung need to withstandthis stress and need to be replaced in case of cell death by a pool ofprogenitor cells.

Two human pathologies stand out by failure of this system.

-   -   1. Bronchopulmonary dysplasia (BPD): Lungs of infants, which are        born prematurely, are more susceptible to lung injury such as        mechanical stress. Also, lungs of premature infants surviving        lung injury often heal with significant scarring or        bronchopulmonary dysplasia (BPD). These lungs have a limited or        underdeveloped regenerative potential.    -   2. Chronic obstructive pulmonary disease (COPD): Lungs of adult        COPD patients seem to respond inappropriately to noxious stimuli        such as smoking. Although they develop a barrier against these        stimuli, their lack of regenerative capabilities in case of cell        death leads to deformation of lung airways and blood vessels.        Both BPD and COPD lead to significant morbidity and mortality.

SUMMARY OF THE INVENTION

The present invention stems from the finding that two genes playimportant roles in tissue development and cancer biology. In particular,the inventors have discovered that these two genes are expressed inpulmonary cells and are required for late branching morphogenesis ofpulmonary epithelium and endothelium and support thedevelopment/maintenance of the refined three dimensional architecture ofthe lung. These genes are essential in the squamous differentiationprogram and development/maintenance of the progenitor (Krt14 expressing)cell pool. Moreover, the inventors have identified crucial roles forthese genes in cancer biology, particularly processes associated withthe acquisition of an immortal and metastatic phenotype (includingcancer progression and metastasis) and cardiac development.

The inventors have ascertained the sequences of the murine and humanforms of these genes. In view of their co-ordinated expression andfunction as regenerative genes for respiratory cells, the inventors havedesignated these genes R2R¹ and R2R². For simplicity, the bulk of thisspecification will use the term “R2R^(1/2)” which is intended torepresent the longer phrase “R2R¹ and/or R2R²”

Accordingly, references to the R2R^(1/2) are to be understood asencompassing all mammalian forms of these genes including, for example,human and rodent (mouse, rabbit, guinea pig, rat etc) forms.Furthermore, in addition to encompassing the entire R2R¹ and/or R2R²gene sequences, it is to be understood that these designations alsoencompass fragments, portions, mutants, derivatives and/orhomologous/orthologues of any of the genes described herein. In thisregard, it should be understood that the term “R2R¹” encompasses themurine sequence encoded by the cDNA sequences designated 2200001115Rikor RIKEN cDNA 2200001I15 as well as the seven human homologuesdesignated FAM25A, FAM25B, FAM25C, FAM25D, FAM25E, FAM25G and FAM25HP.

In addition, where appropriate, the term “R2R^(1/2)” encompasses theproteinaceous products of the R2R¹ and/or R2R² genes or fragments orportions thereof.

In particular, the term “R2R^(1/2)” or indeed either of the terms “R2R¹”or “R2R²”, encompass the sequences given as SEQ ID NOS: 1-12 below orfragments, portions, analogues, variants or derivatives thereof.

The sequence of an exemplary transcript of the murine R2R¹ gene is givenbelow as SEQ ID NO: 1

SEQ ID NO: 1 acactgacacggaccgaaggagtggaaaaagctttacctgtcactgtctgctgccatacgATGCTGGGAGGCCTGGGGAAGCTGGCGGCCGAGGGCCTGGCCCACCGCACAGAGAAAGCCACTGGGGGAGCAGTTCACGCAGTGGAAGAGGTGGTGAGCGAGGTGGTGGGCCACGCCAAGGAGGTTGGAGAGAAGACCATTAATGACGCCCTAAAGAAAGCCCAAGAATCAGGAGACAGGGTGGTGAAGGAGGTCACTGAGAAGGTCACCCACACCATCACTGATGCTGTTACCCATGCGGCAGAAGGCCTGGGAAGACTGGGACAGtgagcctgcctaccagcatggctggcccttcctgaaggtcaataaagagtgtgaaacgtgaaaaaaaaaaaaaaaataacaaaaaaaaaaaaaaaaaa

The coding or translated part of this sequence is underlined andcomprises some 267 nucleotides. This particular portion of SEQ ID NO: 1has been designated SEQ ID NO: 2.

One of skill in this field will appreciate that the 267 translatednucleotides yield a protein comprising 89 amino acids and having thefollowing sequence (designated SEQ ID NO: 3)

SEQ ID NO: 3 MLGGLGKLAAEGLAHRTEKATGGAVHAVEEVVSEVVGHAKEVGEKTINDALKKAQESGDRVVKEVTEKVTHTITDAVTHAAEGLGRLGQ

In addition, the inventors have ascertained the complete sequence of anexemplary human transcript of the R2R¹ gene and this is given as SEQ IDNO: 4 below:

SEQ ID NO: 4 actgtctgctgccacacgATGCTGGGAGGCCTGGGGAAGCTGGCTGCCGAAGGCCTGGCCCACCGCACCGAGAAGGCCACCGAGGGAGCCATTCATGCCGTGGAAGAAGTGGTGAAGGAGGTGGTGGGACACGCCAAGGAGACTGGAGAGAAAGCCATTGCTGAAGCCATAAAGAAAGCCCAAGAGTCAGGGGACAAAAAGATGAAGGAAATCACTGAGACAGTGACCAACACAGTCACAAATGCCATCACCCATGCAGCAGAGAGTCTGGACAAACTTGGACAGtgagtgcacctgctaccacggcccttccccagtctcaataaaaagccatgacatgtg

The coding or translated part of this sequence is underlined andcomprises some 267 nucleotides. This particular portion of SEQ ID NO: 4has been designated SEQ ID NO: 5.

One of skill in this field will appreciate that the 267 translatednucleotides yield a protein comprising 89 amino acids and having thefollowing sequence (designated SEQ ID NO: 6)

SEQ ID NO: 6 MLGGLGKLAAEGLAHRTEKATEGAIHAVEEVVKEVVGHAKETGEKAIAEAIKKAQESGDKKMKEITETVTNTVTNAITHAAESLDKLGQ

The sequence of an exemplary transcript of the murine R2R² gene is givenbelow as SEQ ID NO: 7

SEQ ID NO: 7 GtgactggctgctgtctctagttgttgaggcctcttgggatctyggcgctmacmccwtgctytagwgactccgatagctcccrmggctccagtgsasmcctcggkcggnggnagggaaaaggcacttgctggtagctctgctcacccgcactgggacctggagctggaggactaagaagacagacggctgctgcttgccacagcctggaccATGGACCCCCATGAGATGGTTGTGAAGAATCCATATGCCCACATCAGCATTCCTCGGGCTCACCTGCGCTCTGACCTGGGGCAGCAGTTAGAGGAGGTTCCTTCTTCATCTTCCTCCTCTGAGACTCAGCCTCTGCCTGCAGGAACATGTATCCCAGAGCCAGTGGGCCTCTTACAAACTACTGAAGCCCCTGGGCCCAAAGGTATCAAGGGCATCAAGGGTACTGCTCCTGAGCACGGCCAGCAGACCTGGCAGTCACCCTGCAATCCCTATAGCAGTGGGCAACGTCCATCGGGACTGACTTATGCTGGCCTGCCACCTGTAGGGCGTGGTGATGACATTGCCCACCACTGCTGCTGCTGCCCTTGCTGCTCCTGCTGCCACTGTCCTCGCTTCTGCCGTTGTCACAGCTGTTGTGTTATCTCCtagctgactattgaacctccagggctgtgcagcccaggttcctgctcaatgccaaagtgttgctggacatcaggagcagccgttgtcatgagcatcagccatttcctgccctgagcaggggagcctgtccaccagcgttcagctgtagccttctggaatagggttccagccactagccatgttggcaacaacagggacacccttcacgtcctgcaagactttggcaataaagcaggatgagcgttgctgnncctgntgaaaanaaamwaaawacwgccgttgtcacarcygttrtgttatctmmkagstgacwattgtaammtycagrgctgtrmagcccrggkksckgctcaatgccaaagtgttgmtgsmcmtcrggrgsrgccaagctttacgcggtacccgggattttttttgtacaaaaaggggccccctattagg

The coding or translated part of this sequence is underlined andcomprises some 426 nucleotides. This particular portion of SEQ ID NO: 7has been designated SEQ ID NO: 8.

One of skill in this field will appreciate that the 426 translatednucleotides yield a protein comprising 142 amino acids and having thefollowing sequence (designated SEQ ID NO: 9)

SEQ ID NO: 9 MDPHEMVVKNPYAHISIPRAHLRSDLGQQLEEVPSSSSSSETQPLPAGTCIPEPVGLLQTTEAPGPKGIKGIKGTAPEHGQQTWQSPCNPYSSGQRPSGLTYAGLPPVGRGDDIAHHCCCCPCCSCCHCPRFCRCHSCCVIS

In addition, the inventors have ascertained the complete sequence of anexemplary human transcript of the R2R² gene and this is given as SEQ IDNO: 10 below:

SEQ ID NO: 10 cttgaacccgggaggcagaggttgcagtgagccgagatcgcgcagctgcactccagcctgggcaacagagcaagactccatctcagaaaagaagcagaaagcctccaagagccaatggctctcaagcatcttggtctctgctaagaagaggctcagaggcttagaagccctgcctcgccggggctttgaggtgtgtgagcaatggctggggactgcaggcccgggaatctgagggcctcaccccacttcctttccagagccgtgacctcaggctcacctcctgccctcctctcaggcaagctgcagatgccctttagggcccaggccatgccccggatgtgaggggctgagtcactggtttggcagtgcccctcagagcccaggcctgggctgccacccacctgaggacgagggctgggccagctgtcgtgctccagttgctggggcctcttgggatcttgggaaccccatctctgagccccgccccATGGCCCCGCCCCTCCCAAGGAGGGAAAAGGCGGCTGCCAGTCGCTCAACTCAGGCACTGGGACCTAGAGCTCAGAAGACCGAGAGGACAGACTGCCGTGTTGCCACCACAGGCTGGACCATGGACCCCCAAGAGATGGTCGTCAAGAACCCATATGCCCACATCAGCATCCCCCGGGCTCACCTGCGGCCTGACCTGGGGCAGCAGTTAGAGGTGGCTTCCACCTGTTCCTCATCCTCGGAGATGCAGCCCCTGCCAGTGGGGCCCTGTGCCCCAGAGCCAACCCACCTCTTGCAGCCGACCGAGGTCCCAGGGCCCAAGGGCGCCAAGGGTAACCAGGGGGCTGCCCCCATCCAGAACCAGCAGGCCTGGCAGCAGCCTGGCAACCCCTACAGCAGCAGTCAGCGCCAGGCCGGACTGACCTACGCTGGCCCTCCGCCCGCGGGGCGCGGGGATGACATCGCCCACCACTGCTGCTGCTGCCCCTGCTGCCACTGCTGCCACTGCCCCCCCTTCTGCCGCTGCCACAGCTGCTGCTGCTGTGTCATCTCCtagcccagcccaccctgccagggccaggacccagacttcagcaaatgtggctcacacagtgccgggacatgccgggacatgcggggtggctgttgtcatgggcgtctgccccttcacaccaggcactggggctcagacccaccaggaaggtggccgttcagcccgagctcctgaaacggaatcccaggtcctggctggagagggacacccctgattaccttaaggcccaggcaat aaagcagggtgatcttc

The coding or translated part of this sequence is underlined andcomprises some 552 nucleotides. This particular portion of SEQ ID NO: 10has been designated SEQ ID NO: 11.

One of skill in this field will appreciate that the 552 translatednucleotides yield a protein comprising 184 amino acids and having thefollowing sequence (designated SEQ ID NO: 12)

SEQ ID NO: 12 MAPPLPRREKAAASRSTQALGPRAQKTERTDCRVATTGWTMDPQEMVVKNPYAHISIPRAHLRPDLGQQLEVASTCSSSSEMQPLPVGPCAPEPTHLLQPTEVPGPKGAKGNQGAAPIQNQQAWQQPGNPYSSSQRQAGLTYAGPPPAGRGDDIAHHCCCCPCCHCCHCPPFCRCHSCCCCVIS

As such, the present invention relates to the genes encoded by thesequences designated as SEQ ID NOS: 1, 2, 4, 5, 7, 8, 10 and 11, as wellas any fragments, portions, mutants, variants, derivatives and/orhomologues/orthologues thereof. Typically, fragments, portions, mutants,variants, derivatives and/or homologues/orthologues are functional oractive—that is, they retain the function of the wild type R2R^(1/2)genes.

The term “mutants” may encompass naturally occurring mutants or thoseartificially created by the introduction of one or more nucleic acidadditions, deletions, substitutions or inversions.

One of skill in this field will readily understand that genes homologousto the human and murine R2R^(1/2) genes detailed above may be found in anumber of different species, including, for example, other mammalianspecies. Homologous genes may exhibit as little as approximately 20 or30% sequence homology or identity however, in other cases, homologousgenes may exhibit at least 40, 50, 60, 65 70, 75, 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98, 99% homology to the various nucleotide sequencesgiven above. As such, homologous genes from other species are to beincluded within the scope of this invention.

Using the various nucleic acid and amino acid sequences describedherein, one of skill in the art could readily identify related sequencesin other species, such as other mammals etc. For example, nucleic acidobtained from a particular species may be probed using the probesdescribed herein, for homologous or closely related sequences.

In addition, it should be understood that the present invention alsorelates to the products of the genes encompassed by this invention andin particular the peptides encoded by SEQ ID NOS: 3, 6, 9 and 12.Furthermore, fragments, portions, analogues, variants, derivatives ofany of these or homologous and/or identical proteins are also within thescope of this invention. Typically, fragments, portions, derivatives,variants and/or homologues are functional or active—that is they retainthe function of the wild type R2R^(1/2) protein.

In addition, proteins, polypeptides/peptides homologous/identical to anyof the proteins encoded by SEQ ID NOS: 3, 6, 9 and 12 are also withinthe scope of this invention. Protein or polypeptide/peptide sequenceswhich are considered as homologous or identical to any of the sequencesdescribed herein may exhibit as little as 20% or 30% sequenceidentity/homology. However, homologous/identical sequences may be atleast 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%homologous or identical. Insofar as the invention relates to fragmentsof any of the protein or polypeptide/peptide sequences described herein,it should be understood that a fragment may comprise anywhere betweenabout 10 and n−1 amino acids (where “n” is the number of amino acids inthe complete sequence). For example, a fragment may comprise about 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or about 125 aminoacids (the maximum number of amino acids being determined by the numberof amino acids in the complete sequence)—such fragments/portions may bereferred to as peptide fragments. In one embodiment, the peptidefragments may be antigenic and/or immunogenic—that is to say, theyretain the ability to bind antibodies exhibiting specificity, affinityand/or selectivity for the native (complete) antigen—such as thoseencoded by SEQ ID NOs: 3, 6, 9 and 12.

One of skill in this field will readily understand that for the variousnucleic acid sequences and polypeptides described herein, naturalvariations due to, for example, polymorphisms, may exist betweenR2R^(1/2) genes and proteins isolated from any given species. Inaddition, it is well known in the art, that the degeneracy of thegenetic code permits substitution of one or more bases in a codonwithout alteration to the primary amino acid sequence. As such, geneticdegeneracy may be exploited in order to yield variant nucleic acidsequences which encode peptide or protein sequences substantiallyidentical to the antigen sequences described herein. Indeed, variantsequences provided by this invention may manifest as proteins and/orgenes that exhibit one or more amino acid/nucleic acid substitutions,additions, deletions and/or inversions relative to a reference sequence(for example any of the sequences described above).

As such, it is to be understood that all such variants, especially thosethat are functional or display the desired activity, are to be includedwithin the scope of this invention.

In other embodiment, the invention relates to derivatives of any of theR2R^(1/2) sequences described herein. The term “derivatives” mayencompass R2R^(1/2) gene or peptide sequences which, relative to thosedescribed herein, comprise one or more amino acid substitutions,deletions, additions and/or inversions.

Additionally, or alternatively, analogues of the various peptidesdescribed herein may be produced by introducing one or more conservativeamino acid substitutions into the primary sequence. One of skill in thisfield will understand that the term “conservative substitution” isintended to embrace the act of replacing one or more amino acids of aprotein or peptide with an alternate amino acid with similar propertiesand which does not substantially alter the physcio-chemical propertiesand/or structure or function of the native (or wild type) protein.Analogues of this type are also encompassed with the scope of thisinvention.

As is well known in the art, the degeneracy of the genetic code permitssubstitution of one or more bases in a codon without changing theprimary amino acid sequence. Consequently, although the sequencesdescribed in this application are known to encode the R2R^(1/2) proteinsdescribed herein, the degeneracy of the code may be exploited to yieldvariant nucleic acid sequences which encode the same primary amino acidsequences.

As stated, the present inventors have discovered that the R2R^(1/2)genes (and their protein products) are involved in pulmonary and cardiacmorphogenesis events (such as cell signalling/transition etc.) andsupport the development of the three dimensional architecture of thelung and heart. As such, a first aspect of this invention provides theR2R^(1/2) genes and/or R²R^(1/2) proteins, for use as medicaments or foruse in treating diseases affecting cell/tissue development/structure,differentiation, proliferation and/or morphogenesis. For example, theR2R^(1/2) genes and/or R2R^(1/2) proteins may be for use in treating,for example pulmonary and/or cardiac diseases and/or conditions as wellas cancer—particularly cancers affecting tissues of the pulmonary orcardiac system.

In certain embodiments, the invention may provide the R2R^(1/2) genesand/or R2R^(1/2) proteins for use in modulating cell transition eventssuch as, for example, mesenchymal to epithelial transition (MET) eventsand events involved in the reverse process, epithelial to mesenchymaltransition (EMT).

In a second aspect, the present invention provides the R2R^(1/2) genesand/or R2R^(1/2) proteins for use in treating pulmonary diseases and/orconditions or their use in the manufacture of a medicament for thetreatment of pulmonary diseases and/or conditions.

In a further aspect, the invention provides the R2R¹ gene and/or proteinfor use in treating cardiac diseases. In one embodiment, the R2R¹ geneand/or protein may be used to treat diseases affecting thedevelopment/structure, differentiation, proliferation and/ormorphogenesis of cardiac cells/tissues. In one embodiment, the inventionmay provide the R2R¹ gene and/or protein for use in treating diseasesand/or conditions affecting the development and/or formation of theventricular septum.

In a further aspect, the invention provides the R2R¹ gene and/or proteinfor use in treating cancer. In one embodiment, the R2R¹ gene and/orprotein may be used to treat cancers affecting a variety of tissuesincluding, for example, pulmonary and/or cardiac tissue. More generally,the invention may extend to the treatment of any cancer involvingaberrant/defective MET/EMT processes. Examples of at least some of thecancers which may be treated using the genes and/or proteins of theinvention are detailed below.

It is reiterated that the terms “R2R¹”, “R2R²” and “R2R^(1/2)” encompassnot only the complete gene/peptide sequences as described above, butalso fragments, analogues, homologues, orthologues, variants andderivatives thereof.

The terms “pulmonary disease” or “pulmonary condition” may includepathologies of the lung such as those affecting lung development or the3D architecture of pulmonary tissues. For example, “pulmonary diseases”may include diseases and/or conditions affecting the epithelial cellslining the airways of the lung, the endothelial cells of the pulmonaryvessel network and/or the differentiation and/or growth of these cells.As such, pulmonary diseases may include diseases affecting pulmonarymorphogenesis pathways and events. Pulmonary diseases and/or conditionsaffecting the differentiation of certain pulmonary cell types (forexample the squamous epithelial cells) or the generation and/ormaintenance of progenitor (basal) cell populations may also be treatedwith the compounds, medicaments and methods described herein. By way ofspecific example, diseases such as Bronchopulmonary Dysplasia (BPD)and/or Chronic obstructive pulmonary disease (COPD) may be treated usingthe compounds described herein. In other embodiments, the terms“pulmonary disease” or “pulmonary condition” may include cellproliferation or neoplastic disorders such as cancer, including, forexample, non-small cell lung cancer (NSCLC) and/or small cell lungcancer (SCLC).

The terms “cardiac disease” or “cardiac condition” may includepathologies of the heart such as those affecting cardiac development orthe 3D architecture of cardic tissues. Cardiac diseases may includediseases affecting cardiac morphogenesis pathways and events, inparticular mesenchymal to epithelial and epithelial to mesenchymaltransition. By way of specific example, diseases such as atrial andventricular septal defects, atrioventricular canal defects, malformationof cardiac (atrioventricular) valves and coronary arteries may betreated using the compounds described herein.

It should also be understood that since the R2R^(1/2) genes/proteinsdescribed herein have been shown to play a role in morphogenesis events(such as cell signalling etc.) and support the development of the threedimensional architecture of complex tissues such as the lung and/orheart, they may find application in regenerative medicine. By way ofexample, where stem cells (for example adult, embryionic or reprogrammedsomatic cells (such as iPS cells) are used to repair or reconstruct, forexample, damaged or diseased tissue, the proteins and/or genes providedby this invention may be used to facilitate tissue development.

The R2R^(1/2) protein and genes play an important role in themorphogenesis events of the lung and heart as gatekeepers of epithelialto mesenchymal and mesenchymal to epithelial transition, Therefore, theymay find application in cancer biology and cancer treatment. By way ofexample, diseases such as transformation of localized cancer to cancermetastasis may be treated using compounds described herein.

In addition to providing various uses and medicaments involving theR2R^(1/2) genes and/or proteins described herein, the present inventionalso provides methods of treating subjects suffering from any of thediseases and/or conditions described herein, including any of thecardiac/pulmonary diseases and/or conditions outlined above. As such athird aspect of this invention provides a method of treating acardiac/pulmonary disease and/or condition comprising the steps ofadministering to a subject in need thereof, a therapeutically effectiveamount of the R2R¹ and/or R2R² genes and/or R2R¹ and/or R2R² proteinsdescribed herein.

Where a cardiac or pulmonary disease or condition results from, or isassociated with, a lack of or defective, R2R^(1/2) gene and/or proteinexpression/function, the use of a functional R2R^(1/2) gene or proteinas described herein, may provide a means of restoring normal (or wildtype) R2R^(1/2) gene/protein function so as to treat and/or alleviatingthe symptoms of the disease or condition.

It will be appreciated that the uses, medicaments and methods oftreatment described herein may require the generation of recombinantR2R^(1/2) genes/proteins and as such, the present invention furthercontemplates methods of generating and/or expressing recombinantR2R^(1/2) genes and/or proteins. One of skill in this field willappreciate that PCR techniques may be exploited to selectively obtainR2R^(1/2) gene sequences from a variety of sources including, forexample, lung tissue. These sequences may be ligated to variousexpression or regulatory control sequences such as, for example,promoter, operator, inducer, enhancer and/or silencer elements, ribosomebinding sites and/or terminator sequences. Suitable regulatory orexpression control sequences may be selected by those skilled in thisfield for any given host. In a further embodiment, the PCR derivedR2R^(1/2) gene sequences may be introduced into a vector (such as aplasmid or expression cassette). In one embodiment, the vector mayfurther comprise a nucleotide sequence of a tag or label to assist inprotein purification procedures.

A host cell may be transformed with the vector and maintained underconditions suitable to induce expression of the R2R^(1/2) gene sequenceand production of recombinant R2R^(1/2). Vectors into which R2R^(1/2)gene sequences (or fragments thereof) have been cloned, may beintroduced or transfected into cells using a variety of techniques—suchtechniques may otherwise be referred to as transfection protocols.Transfection protocols utilise conditions which render cell membranespermeable to compounds such as nucleic acids. By way of example, it maybe possible to facilitate the transfection of vectors, includingexpression vectors, into cells using electroporation, heat shock,chemical compounds such, for example, calcium phosphate, stronitiumphosphate, microinjection techniques and/or gene guns.

Techniques used to purify recombinant proteins generated in this way areknown and, where the recombinant protein is tagged or labelled, thesemay include the use of, for example, affinity chromatography techniques.

In view of the above, the fourth and fifth aspects of this inventionprovide an expression vector comprising an R2R^(1/2) gene sequence and ahost cell transformed therewith, respectively.

In addition to providing the R2R^(1/2) genes and/or proteins as a meansof treating various diseases and/or conditions (for example cardiacand/or pulmonary diseases and/or conditions), the present invention alsoprovides compounds capable of modulating the expression of the R2R^(1/2)genes and which may be useful in the treatment of conditions that resultfrom, or are associated with R2R^(1/2) gene/protein over-expression.Such compounds may be oligonucleotides, preferably antisenseoligonucleotides which may take the form of, for example DNA and/or RNA.In one embodiment, the oligonucleotides are RNA molecules known to thoseskilled in this field as small/short interfering and/or silencing RNAand which will be referred to hereinafter as siRNA. Such siRNAoligonucleotides may take the form of native RNA duplexes or duplexesthat have been modified in some way (for example by chemicalmodification) to be nuclease resistant. Additionally, or alternatively,the siRNA oligonucleotides may take the form of short hairpin RNA(shRNA) expression or plasmid constructs that correspond to, orcomprise, the siRNAs described herein.

The oligonucleotides provided by this invention may be designed tomodulate the expression the R2R^(1/2) genes. By analysing native orwild-type R2R^(1/2) sequences and with the aid of algorithms such asBIOPREDsi, one of skill in the art could easily determine orcomputationally predict nucleic acid sequences that have an optimalknockdown effect for these genes (see for example:http://www.biopredsi.org/start.html). Accordingly, the skilled man maygenerate and test an array or library of different oligonucleotides todetermine whether or not they are capable of modulating the expressionof the R2R^(1/2) genes.

In view of the above, the antisense oligonulcoeotides and/or siRNAmolecules described herein may be used (i) treat any of the diseasesand/or conditions described herein—in particular (ii) to treat pulmonarydiseases and/or disorders, (iii) to treat cardiac diseases and/orconditions and (iv) to treat cancer. Furthermore, the antisenseoligonulcoeotides and/or siRNA molecules described herein may be used inthe manufacture of medicaments for treating the diseases outlined as(i)-(iv) above or in methods of treating subjects suffereing from suchdiseases and/or disorders.

In addition, antibodies (or antigen binding fragments thereof) capableof binding to the R2R^(1/2) proteins may be useful in the treatment ofthe diseases and/or conditions described herein, including, for example,cardiac and/or pulmonary diseases and/or conditions. Antibodies whichblock or neutralise the function of the R2R^(1/2) proteins may beparticularly useful where a disease and/or condition results from overexpression of an R2R^(1/2) protein. The techniques used to generatemonoclonal antibodies (mAbs) are well known and can easily be exploitedto generate mAbs specific for either of the R2R¹ and/or R2R² proteins orfragments thereof. Similalry, the processes used to generate polyclonalantibodies are also well established and may be used to generateantibodies specific for either of the R2R¹ and/or R2R² proteins orfragments thereof.

Other compounds useful in the treatment of diseases and/or conditionsdescribed herein (for example cardiac and/or pulmonary conditions and/ordisorders or cancer) may include for example, proteins, peptides, aminoacids, carbohydrates and other small organic molecules.

In addition to the above, isolated R2R^(1/2) nucleotide and/or proteinsequences may be used as the basis for the design of probes and/orprimers for use in ex vivo and/or in situ detection and expressionstudies. Typical detection studies include, for example, polymerasechain reaction (PCR), hybridisation studies, sequencing protocols andimmunological and/or Southern/Northern blotting detection techniques.

In principle any polynucleotide (or oligonucleotides) or polypeptidefragment designed from the sequences described above may be used in saiddetection and/or expression studies.

Typically, polynucleotide fragments for use as probes and/or primers,will comprise 10-30 nucleotides (although other lengths may be usefulfor certain applications) and exhibit a degree of specificity for aparticular sequence and will not bind unrelated sequences. Similarly,polypeptide fragments to be used as probes may also be relatively shortand typically may comprise 5-20 amino acids (although other slightlyshorter or longer lengths may be useful for some applications).

It will be readily understood that careful selection of the primer/probesequence and the use of stringent (preferably highly stringent)hybridisation conditions will minimise any non-selective binding.

Accordingly, oligonucleotides probe and/or primer sequences having atleast 50%, at least 75%, at least 90% or at least 95% complementarity aswell as those having exact (i.e. 100%) complementarity to all or part ofthe nucleotide sequences described herein are to be considered asencompassed within this invention.

Hybridisation between a probe/primer and a nucleic acid sequence (suchas any described herein) may be effected at a temperature of about 40°C.-75° C. in 2-6×SSC (i.e. 2-6×NaCl 17.5 g/l and sodium citrate (SC) at8.8 g/l) buffered saline containing 0.1% sodium dodecyl sulphate (SDS).Of course, depending on the degree of similarity between theprobe/primer and the sequence, buffers with a reduced SSC concentration(i.e. 1×SSC containing 0.1% SDS, 0.5×SSC containing 0.1% SDS and 0.1×SSCcontaining 0.1% SDS).

Polypeptide probes having at least 30%, 50%, 70%, 75%, 80%, 85%, 90% or95% identity as well as those having exact (i.e. 100% identity) to allor part of the amino acid sequences disclosed herein, are to beconsidered as within the scope of this invention.

As such, a further aspect of this invention provides oligonucleotidesprobes and/or primers designed to hybridise to all or part of a sequenceselected from the group consisting of SEQ ID NOS: 1; 2; 4; 5; 7; 8; 10and 11. In addition, a further aspect provides polypeptide probesdesigned to bind to all or part of a sequence selected from the groupconsisting of SEQ ID NOS: 3; 6; 9 and 12

In another aspect the present invention provides a method of diagnosinga pulmonary disease or condition and/or susceptibility thereto, whereinthe method comprises determining if the R2R¹ and/or R2R² genes in asubject are aberrantly expressed.

Subjects diagnosed as suffering from, for example cancer and/or acardiac and/or pulmonary disease and/or condition may exhibit aberrant(i.e. increased or decreased) R2R¹ and/or R2R² gene/protein expression.The term “aberrant expression” should be understood to encompass levelsof gene expression that are either increased and/or decreased relativeto the expression observed in sample derived from a healthy subject or asubject who is not suffering from the diseases and/or condition (namelya cardiac or pulmonary disease and/or condition or cancer).

The term “sample” should be understood as including samples of bodilyfluids such as whole blood, plasma, serum, saliva, sweat and/or semen.In other instances “samples” such as tissue biopsies and/or scrapingsmay be used. In particular lung tissue biopsies and/or scrapings may beused. In addition, a sample may comprise a tissue or gland secretion andwashing protocols may be used to obtain sample of fluid secreted into,for example, the lung. Suitable washing protocols may includebroncho-alveolar lavage procedures. One of the skill in this field willappreciate that the samples described above may yield quantities ofR2R^(1/2) nucleic acid (i.e. DNA or RNA) and/or R2R^(1/2) proteins,peptides (or fragments thereof). Furthermore, these methods may comprisethe first step of providing a sample from a subject suspected ofsuffering from a pulmonary disease and/or condition or who may be atrisk of developing a pulmonary disease and/or condition.

An increase in the level of R2R¹ and/or R2R² gene/protein expression maybe associated with any of the diseases and/or conditions described aboveor susceptibility thereto. For example an increase in R2R^(1/2)gene/protein expression may be indicative of excessive cellularproliferation and/or differentiation—and may be associated with, forexample, a neoplastic condition such as cancer (i.e, lung cancer).Decreases in R2R¹ and/or R2R² gene/protein expression may be indicativeof pathological conditions characterised by poor or impairedcardiac/lung development. Such conditions may result in tissue injury(due to mechanical stresses acting within the heart/lung), scarring,loss of structural integrity of the heart/lung and deformed or damagedlung airways or cardiac structure.

One of skill in the art will be familiar with the techniques that may beused to identify levels of proteins and/or genes, such as the R2R^(1/2)genes and/or proteins, in samples such as those listed above.

Such techniques may include, for example, polymerase chain reaction(PCR) based techniques such as real-time PCR (otherwise known asquantitative PCR). In the present case, real time-PCR may used todetermine the level of expression of the genes encoding the R2R¹ and/orR2R² proteins. Typically, and in order to quantify the level ofexpression of a particular nucleic acid sequence, reverse transcriptasePCR may be used to reverse transcribe the relevant mRNA to complementaryDNA (cDNA). Preferably, the reverse transcriptase protocol may useprimers designed to specifically amplify an mRNA sequence of interest.Thereafter, PCR may be used to amplify the cDNA generated by reversetranscription.

Typically, the cDNA is amplified using primers designed to specificallyhybridise with a certain sequence and the nucleotides used for PCR maybe labelled with fluorescent or radiolabelled compounds.

One of skill in the art will be familiar with the technique of usinglabelled nucleotides to allow quantification of the amount of DNAproduced during a PCR. Briefly, and by way of example, the amount oflabelled amplified nucleic acid may be determined by monitoring theamount of incorporated labelled nucleotide during the cycling of thePCR.

Further information regarding the PCR based techniques described hereinmay be found in, for example, PCR Primer: A Laboratory Manual, SecondEdition Edited by Carl W. Dieffenbach & Gabriela S. Dveksler: ColdSpring Harbour Laboratory Press and Molecular Cloning: A LaboratoryManual by Joseph Sambrook & David Russell: Cold Spring HarbourLaboratory Press.

Other techniques that may be used to determine the level of R2R¹ and/orR2R² gene expression in a sample include, for example, Northern and/orSouthern Blot techniques. A Northern blot may be used to determine theamount of a particular mRNA present in a sample and as such, could beused to determine the amount of R2R¹ and/or R2R² gene expression.Briefly, mRNA may be extracted from, for example, a cell based or cellfree system modified to include expressible R2R¹ and/or R2R² genes usingtechniques known to the skilled artisan, and subjected toelectrophoresis. A nucleic acid probe, designed to hybridise (i.e.complementary to) an mRNA sequence of interest—in this case the mRNAencoding the R2R¹ and/or R2R² proteins, may then be used to detect andquantify the amount of a particular mRNA present in a sample.

Additionally, or alternatively, a level of R2R¹ and/or R2R² geneexpression may be identified by way of microarray analysis. Such amethod would involve the use of a DNA microarray that comprises nucleicacid derived from the R2R¹ and/or R2R² genes. To identify the level ofR2R¹ and/or R2R² gene expression, one of skill in the art may extractthe nucleic acid, preferably the mRNA from a system (either cell basedor cell free) subjected to the method described in the first aspect ofthis invention, and subject it to an amplification protocol such as,reverse transcriptase PCR to generate cDNA. Preferably, primers specificfor a certain mRNA sequence—in this case the sequences encoding the R2R¹and/or R2R² genes may be used.

The amplified R2R¹ and/or R2R² cDNA may be subjected to a furtheramplification step, optionally in the presence of labelled nucleotides(as described above). Thereafter, the optionally labelled amplified cDNAmay be contacted with the microarray under conditions that permitbinding with the DNA of the microarray. In this way, it may be possibleto identify a level of R2R¹ and/or R2R² gene expression.

Further information regarding the above described techniques may befound in, for example, PCR Primer: A Laboratory Manual, Second EditionEdited by Carl W. Dieffenbach & Gabriela S. Dveksler: Cold SpringHarbour Laboratory Press and Molecular Cloning. A Laboratory Manual byJoseph Sambrook & David Russell: Cold Spring Harbour Laboratory Press.

In order to determine the level of R2R^(1/2) proteins in a sample,immunological techniques exploiting agents capable of binding R2R^(1/2)proteins, may be used.

In one embodiment, the diagnostic methods described above may comprisethe step of contacting a substrate (or portion thereof) with a sample tobe tested, under conditions which permit the association, interaction,binding and/or immobilization of any R2R^(1/2) protein present in thesample, to said substrate.

Suitable substrates may include, for example, glass, nitrocellulose,paper, agarose and/or plastics. A substrate such as, for example, aplastic material, may take the form of a microtitre plate.

Alternatively, the substrate to be contacted with the sample to betested may comprise an agent capable of binding R2R^(1/2) protein(s).Preferably, the agent capable of binding the R2R^(1/2) proteins is/arebound to the substrate (or at least a portion thereof). Suitable bindingagents may include, for example, antibodies such as monoclonal orpolyclonal antibodies and/or other types of peptide or small moleculecapable of binding to the R2R^(1/2) proteins. It is to be understoodthat this definition applies to all types of binding agent mentionedherein. As such, the substrate (or a portion thereof) may be contactedwith the sample to be tested under conditions that permit binding orinteraction between the agents capable of binding the R2R^(1/2) proteinand any R2R^(1/2) protein present in the sample.

Any R2R^(1/2) protein bound to the substrate or agents capable ofbinding the R2R^(1/2) protein(s), may be detected with the use of afurther agent capable of binding the R2R^(1/2) protein(s) (referred tohereinafter as the “primary binding agent”). Additionally, oralternatively, the primary binding agents may have affinity for, or bindto, R2R¹/R2R² protein: substrate complexes or complexes comprising theR2R^(1/2) proteins and the abovementioned agents capable of binding theR2R^(1/2) proteins.

The primary binding agents may be conjugated to moieties which permitthem to be detected (referred to hereinafter as “detectable moieties”).For example, the primary agents may be conjugated to an enzyme capableof reporting a level via a colorimetric chemiluminescent reaction. Suchconjugated enzymes may include but are not limited to Horse RadishPeroxidase (HRP) and Alkaline Phosphatase (AlkP). Additionally, oralternatively, the primary binding agents may be conjugated to afluorescent molecule such as, for example a fluorophore, such as FITC,rhodamine or Texas Red. Other types of molecule that may be conjugatedto binding agents include radiolabelled moieties.

Alternatively, any R2R^(1/2) protein bound to the substrate or agentscapable of binding the R2R^(1/2) proteins, may be detected by means of ayet further binding agent (referred to hereinafter as “secondary bindingagents”) having affinity for the primary binding agents. Preferably, thesecondary binding agents are conjugated to detectable moieties.

The amount of primary binding agent (or secondary binding agent boundthereto) bound to R2R^(1/2) protein(s), may represent the level ofR2R^(1/2) protein(s) present in the sample tested.

In one embodiment, the methods for identifying a level of R2R^(1/2)protein, may take the form of “dip-stick” test, wherein a substrate (orportion thereof) is contacted with a sample to be tested underconditions which permit the binding of any R2R^(1/2) protein(s) presentin the sample to the substrate or a binding agent bound or immobilisedthereto.

In a further embodiment, the methods may take the form of animmunological assay such as, for example, an enzyme-linked immunosorbentassay (ELISA). An ELISA may take the form of a “capture” ELISA wherein,a sample to be tested is contacted with a substrate, and any R2R^(1/2)protein(s) present in the sample is/are “captured” or bound by a bindingagent (capable of binding the R2R^(1/2) proteins) bound or immobilizedto the substrate. Alternatively, the sample may be contacted with thesubstrate under conditions that permit “direct” binding between anyR2R^(1/2) protein(s) present in the sample and the substrate.

Each of the ELISA methods described above may comprise a “direct”protein detection step or an “indirect” identification step. ELISAsinvolving such steps may be known as “direct” ELISAs or “indirect”ELISAs.

A “direct” ELISA may involve contacting the sample to be tested with asubstrate under conditions that permit the binding of any R2R^(1/2)protein(s) present in the sample to the substrate and/or a binding agentbound thereto. After an optional blocking step, bound R2R^(1/2)protein(s) may be detected by way of an agent capable of binding theR2R^(1/2) proteins (i.e. a primary binding agent). Preferably, theprimary binding agents are conjugated to a detectable moiety.

An “indirect” ELISA may comprise the further step of, after contactingthe R2R^(1/2) protein(s) with a primary binding agent, using a furtherbinding agent (secondary binding agent) with affinity or specificity forthe primary binding agent. Preferably, the secondary binding agent maybe conjugated to a detectable moiety.

Other immunological techniques which may be used to identify a level ofR2R^(1/2) protein in a sample include, for example, immunohistochemistrywherein binding agents, such as antibodies capable of binding theR2R^(1/2) protein(s), are contacted with a sample, preferably a tissuesample, under conditions which permit binding between any R2R^(1/2)protein(s) present in the sample and the R2R^(1/2) protein bindingagent. Typically, prior to contacting the sample with the binding agent,the sample is treated with, for example a detergent such as Triton X100.Such a technique may be referred to as “direct” immunohistochemicalstaining.

Alternatively, the sample to be tested may be subjected to an indirectimmunohistochemical staining protocol wherein, after the sample has beencontacted with a R2R^(1/2) protein binding agent, a further bindingagent (a secondary binding agent) which is specific for, has affinityfor, or is capable of binding the R2R^(1/2) protein binding agent, isused to detect R2R^(1/2) protein/binding agent complexes.

The skilled man will understand that in both direct and indirectimmunohistochemical techniques, the binding agent or secondary bindingagent may be conjugated to a detectable moiety. Preferably, the bindingagent or secondary binding agent is conjugated to a moiety capable ofreporting a level of bound binding agent or secondary binding agent, viaa colorimetric chemiluminescent reaction.

In order to identify the levels of R2R^(1/2) protein(s) present in thesample, one may compare the results of an immunohistochemical stain withthe results of an immunohistochemical stain conducted on a referencesample. By way of example, a sample that reveals more or less boundR2R^(1/2) protein binding agent (or secondary binding agent) than in areference sample, may have been provided by a subject with a particulardisease and/or condition.

Other techniques that exploit the use of agents capable of binding theR2R^(1/2) proteins include, for example, techniques such as Western blotor dot blot. A Western blot may involve subjecting a sample toelectrophoresis so as to separate or resolve the components, for examplethe proteinaceous components, of the sample. The resolved components maythen be transferred to a substrate, such as nitrocellulose. In order toidentify any R2R^(1/2) protein(s) present in the sample, the substratemay be contacted with a binding agent capable of binding R2R^(1/2)protein(s) under conditions which permit binding between any R2R^(1/2)protein(s) present in the sample and the agents capable of bindingR2R^(1/2) protein(s).

Advantageously, the agents capable of binding R2R^(1/2) protein(s) maybe conjugated to a detectable moiety.

Alternatively, the substrate may be contacted with a further bindingagent having affinity for the binding agent(s) capable of bindingR2R^(1/2) protein(s). Advantageously, the further binding agent may beconjugated to a detectable moiety.

In the case of a dot blot, the sample or a portion thereof, may becontacted with a substrate such that any R2R^(1/2) protein(s) present inthe sample is bound to or immobilised on the substrate. Identificationof any bound or immobilised R2R^(1/2) protein(s) may be conducted asdescribed above.

In any of the abovementioned techniques, the amount of primary orsecondary binding agent detected is representative of, or proportionalto, the amount of R2R^(1/2) protein present in the sample. Furthermore,the results obtained from any or all of the diagnostic methods describedherein may be compared with the results obtained from reference orcontrol samples derived from healthy subjects known not to be sufferingfrom, or susceptible to, a particular disease or disorder (such as, forexample a cardiac and/or pulmonary disease or disorder and/or cancer).

A further aspect of this invention provides a method of identifying orobtaining agents which modulate the expression of the R2R¹ and/or R2R²genes, said method comprising the steps of contacting the R2R¹ and/orR2R² genes with a test agent and detecting any modulation of R2R^(1/2)gene expression.

One of skill in this field will appreciate that a method such as thatdescribed in this 9th aspect of the invention may be conducted insystems such as, for example, cell based or cell free systems, modifiedto include the R2R¹ and/or R2R² genes. By way of example, cells may betransfected with nucleic acid comprising either the R2R¹ or R2R² gene.In one embodiment, the nucleic acid may take the form of a vector (forexample a plasmid or expression cassette as described above).

In one embodiment, the results obtained from the methods described abovemay be compared to those obtained from a control method in which theR2R¹ and/or R2R² genes have not been contacted with a test agent. Inthis way, it may be possible to determine whether or not said agent iscapable of modulating the expression of the R2R¹ and/or R2R² genes.Where the level of R2R¹ and/or R2R² gene expression is less or greaterthan the level of expression detected in the control method, the testagent may be useful as a modulator of R2R¹ and/or R2R² gene expression.Where the level of expression is the same as that observed in thecontrol methods, the test agent is most likely not capable of modulatingthe expression of the R2R¹ and/or R2R² genes.

Suitable test agents may take the form of nucleic acids, for example theantisense oligonucleotides described above, proteins, peptides, aminoacids, antibodies (and fragments thereof), carbohydrates and other smallorganic molecules.

In further aspect, the present invention provides pharmaceuticalcompositions comprising the R2R^(1/2) genes/proteins described above,antisense oligonucleotides (DNA or RNA) as described herein and/or anyof the agents identified by the methods provided by the 9th aspect ofthis invention and which are capable of modulating the expression orfunction of the R2R¹ and/or R2R² genes/proteins, in association with apharmaceutically acceptable excipient, carrier or diluent. Suchcompositions may find application in, for example, the treatment of thevarious diseases and/or conditions described herein including thecardiac and/or pulmonary diseases and/or cancers described above.

Preferably, the pharmaceutical compositions provided by this inventionare formulated as sterile pharmaceutical compositions. Suitableexcipients, carriers or diluents may include, for example, water,saline, phosphate buffered saline, dextrose, glycerol, ethanol, ionexchangers, alumina, aluminium stearate, lecithin, serum proteins, suchas serum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water salts or electrolytes, such as protaminesulphate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycon,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polypropylene-block polymers, polyethylene glycol and woolfat and the like, or combinations thereof.

Said pharmaceutical formulation may be formulated, for example, in aform suitable for oral, parenteral or topical administration. In oneembodiment, the pharmaceutical composition may be formulated such thatit can be inhaled. Compositions that are to be administered byinhalation may take the form of fine powders or solutions which can beaerosolised and inhaled as droplets. One of skill in this field will befamiliar with devices that may be used to deliver compositions directlyto the lung by, for example, inhalation. The droplet or particle size ofthe composition can be altered such that the drug can access differentregions of the lung. For example, once inhaled, small particles ordroplets may penetrate deep into the lung tissue and in some cases mayreach the alveoli.

Pharmaceutical compositions formulated for topical administration may bepresented as an ointment, solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water liquid emulsion.

Compounds capable of modulating the R2R¹ and/or R2R² genes, such as, forexample, those identified by the methods described herein or theR2R^(1/2) gene/protein fragments, antisense oligonucleotides, andantibodies described herein, may find further application as modulatorsof cell differentiation. As stated, the inventors have determined thatthe R2R¹ and R2R² genes and their products are involved in the pathwaysthat modulate the differentiation of pulmonary epithelial cells,particularly the squamous epithelial cells generated from the pulmonaryepithelial progenitor or basal cell population (i.e. Krt14^(+ve) cells).

Compounds that modulate cell (for example, pulmonary epithelial cell)differentiation may be particularly useful in the treatment of disorderssuch as BPD which yield damaged and scarred lungs with a reduced orunderdeveloped regenerative potential. As such, by administering orusing a compound which modulates cell differentiation, it may bepossible to improve or restore the regenerative potential of the lung.One of skill in this field will readily understand that compoundscapable of enhancing or promoting R2R¹ and/or R2R² gene expression orwhich restore the function of these genes may be particularly useful.

In a further aspect, the present invention provides an animal model forstudying tissue development and/or cell transition events as well ascertain diseases and/or conditions (including cardiac and/or pulmonarydiseases and/or conditions and cancer). In one embodiment, the animalmodel may be generated by manipulating or modulating (i.e. up or downregulating) the expression of the R2R^(1/2) genes/proteins. Additionallyor alternatively, an animal model may be generated by disruptingR2R^(1/2) gene expression. As described above, the disclosure of anumber of human and murine R2R^(1/2) gene/protein sequences hereinensure that the skilled man could readily manipulate/modulate thesesequences in situ to generate animal models. For example, “knock-out”animals may be created, that is, the expression of R2R^(1/2) genesequences is reduced or substantially eliminated. Such models are usefulfor testing the effects of drugs potentially useful in the treatment ofthe diseases and/or disorders described herein and/or for determiningthe function or role of a particular gene. Alternatively, animal modelsin which the expression of the R2R^(1/2) genes/proteins are upregulated,may also be created. In this way, the efficacy and function of drugsaimed at suppressing upregulated R2R^(1/2) gene/protein expression maybe tested. It is also possible to investigate the effect of upregulatedR2R^(1/2) gene protein expression in these animals.

In other embodiments, substitutions, additions, deletions and/orinversions may be introduced into the R2R^(1/2) gene sequences in orderto effect changes in the activity of the proteins and to help elucidatethe function of certain domains, amino acids and the like. Additionally,or alternatively, knockout animals, such as those described above, maybe transformed with any of the gene sequences described herein. This isparticularly useful where the effect of a variant or mutated R2R^(1/2)gene or efficacy of a drug or compound capable of suppressing theexpression or function of said variant or mutated sequence, is to beinvestigated.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in detail with reference tothe following Figures which show:

FIG. 1. Comparison of endothelial and epithelial cells from Vegf+/+ atE16.5. Volcanoplot showing on the y-axis the significance of each genewhen testing for the effect of cellular origin (based on LIMMA). Thex-axis shows the fold change when comparing the 2 tissues. Ker+ cellsexpress archetypical epithelial genes, while il+ cells transcriberepresentative endothelial genes. Endothelial Cldn5 and epithelial Foxa1are highlighted because both were also present as top genes in theY-axis (PC₂) of the unsupervised spectral map analysis (see FIG. 3).

FIG. 2. Schematic representation of the laser capture microdissectionproces. Embryonic thoraces (A) were cut in 8 mm sections and placed onslides (B). Double immunohistochemical staining on these tissue sections(C) with anti-‘pan’ keratin and GS-IB4 isolectin separated pulmonaryairway cells with epithelial (D) distinctiveness from the surroundingcells with endothelial (E) characteristics. Specific cell groupsisolated by laser capture microdissection are shown in F and G. (L=lung,H=heart).

FIG. 3. Spectral map analysis with first principal component (PC1 onX-axis) and second principal component (PC2 on Y-axis). Time effect orembryonic age was uncovered in PC1 (accountable for 35% of the variationin the data set). Gene probes of genes expressed later in embryonic lifeas Sftpc were located further on the X-axis. Difference in cellularorigin (il+ versus ker+ cells) was found in PC2. Gene probes ofepithelial genes (‘EPI’) were located further on the Y-axis in contrastto gene probes of endothelial gene (‘ENDO’) families. The differentsample groups were separated in the spectral map along PC1 (embryonicage) and PC2 (cellular origin). Ker+ samples gathered on the epithelialside of the Y-axis, and il+ samples grouped on the endothelial side. Thesamples also distributed along the X-axis according to their embryonicage. Samples of later embryonic days were located further on the X-axis.The panel below shows 3 expression profiles: (1) showing the differencebetween epithelial and endothelial cells for Foxal expression, (2)showing the effect of embryonic age for Afp expression and (3) showingthe genotype dependent profile over embryonic age for Him expression.

FIG. 4, Comparison of epithelial cells from Vegf+/+ and Vegf120/120genotypes. A. Volcanoplot showing on the y-axis the significance of eachgene for testing whether the expression profile over embryonic agediffers between the Vegf+/+ and Vegf120/120 genotypes (based on LIMMA).The x-axis shows the fold change in induction when comparing the 2genotypes, Vegf+/+ versus Vegf120/120 at E16.5. Upregulation of Krt6a,EDC and SCC genes in wild type ker+ cells is highlighted. B. Airwayepithelial cells in E16.5 Vegf+/+ lungs defined by anti-cytokeratin 4,5, 6, 8, 10, 13, and 18 staining. Distal airway cells develop a flat orsquamous cell morphology (yellow arrow) in contrast to their proximalneighbours (white arrow) C. Allocation of differential expression allongchromosome 3. This highlights the downregulation of the genes in the EDCof Vegf120/120 as compared to wildtype.

FIG. 5A. IF=Intermediate Filament Keratins (red) group anchoring to theDesmosome (orange) containing Dsc1. Pkp1 (purple) links IntermediateKeratin Filaments to Cadherin proteins of the Adherens type junction(yellow). Pkp1 also regulates the protein content of the Desmosome. EDCand SCC cluster genes interact with Intermediate Filament Keratins. Thestar symbol highlights the upregulated genes and gene clusters in theIntermediate Filament network. Upregulation of Eps811 (coding for anactin-capping protein) coordinates Intermediate Filament with Actinremodelling.

B. Volcanoplot showing on the y-axis the significance of each gene fortesting whether the expression profile over embryonic age differsbetween endothelial cells of Vegf+/+ and Vegf120/120 genotypes (based onLIMMA). The x-axis shows the fold change in induction when comparing the2 genotypes, Vegf+/+ versus Vegf120/120 at E16.5. The Krt, Dsc1, Pkp1,EDC and SCC cluster genes are highlighted. The Krt genes upregulated inwild type Vegf+/+ il+ cells differ from the Krt genes expressed in theequivalent wild type ker+ cells. Krt14 and Krt1 are trademark basal cellgenes.

C. Immunofluorescent image (40× magnification) of GS-IB4 isolectinstaining cells (il+ cells) in wild type E16.5 embryonic lungs. Il+ cellsfollow the same architectural pattern of the ker+ cells of the distalairways.

FIG. 6. Volcanoplot based on LIMMA analysis of the group ofanti-cytokeratin 4-5-6-8-10-13-18 staining cells. (Linear Models forMicroarray Data: differences between Vegf120/120 knockout and wild typelittermates in expression profiles over embryonic age were testedthrough a two-way interaction of Vegf genotype and time). The RIKEN cDNA2200001I15 gene and RIKEN cDNA 2310002J15 gene are highlighted.

FIG. 7. Volcanoplot based on LIMMA analysis of the group of GS-IB4binding cells. (Linear Models for Microarray Data: differences betweenVegf120/120 knockout and wild type littermates in expression profilesover embryonic age were tested through a two-way interaction of Vegfgenotype and time). The RIKEN cDNA 2200001I15 gene and RIKEN cDNA2310002J15 gene are highlighted.

FIG. 8: cDNA sequence alignments of the human hR4RA transcript (=humanR2R¹) versus mouse R4Ra transcript (=mouse R2R¹). It should be notedthat the largest contig constructed from sequenced RIKEN cDNA 2200001I15gene clones is referred to as ‘mouse R4Ra transcript (=mouse R2R¹)’.

FIG. 9: Protein sequence alignments of the translated human hR4RA(=human R2R¹) transcript versus translated mouse R4Ra (=mouse R2R¹)transcript.

FIG. 10: cDNA sequence alignments of the human hR4RD transcript (=humanR2R²) versus mouse R4Rd transcript (=mouse R2R²). It should be notedthat the largest contig constructed from sequenced RIKEN cDNA 2310002J15gene clones is referred to as ‘mouse R4Rd transcript (=mouse R2R²)’.

FIG. 11: Protein sequence alignments of the translated human hR4RD(=human R2R²) transcript versus translated mouse R4Rd (=mouse R2R²)transcript.

FIG. 12: VEGF-A (VEGF¹⁶⁴ dependent upregulation of R2R¹ in thedeveloping ventricular septum of the mouse embryo. Red=wild type,blue=vEGF_(120/120) knockout mouse, lacking the VEGF¹⁶⁴ isoform.

FIG. 13: Human R2R² homologue (=C9orf196) expression over time (24-72 h)in human adult primary lung epithelial cells.

FIG. 14: siRNA knockdown of VEGF¹⁶⁵ leads to knockdown of the genesresponsible for the basal cell program and squamous differentiationprogram. siRNA knockdown in human Primary Bronchial Epithelial Cells(PBEC's) reliably models the effect of gene knockdown of genes ofinterest. (a) RTqPCR: siRNA mediated knockdown of VEGFA and VEGF¹⁶⁵expression leads to knockdown of KRT14 (basal cell marker) expression.KRT14 relative expression (PGK1 normalized), VEGFA and VEGF¹⁶⁵ areplotted versus siRNA. Included are two negative controls (called nr 10and 11). siRNA directed against KRT14 expression is included as apositive control. siRNA VEGFA is directed against all VEGFA isoforms.

FIG. 15 A-J: Global gene expression changes brought about by siRNAmediated knockdown of VEGFA in PBEC's. Changes in global gene expressionmatch exactly the changes observed in the Vegf120/120 knockout mouse,Graphs (a)-(j) depict the significance of changes in gene expressionafter administration during 24 h of an siRNA directed against VEGF.Graphs (a)-(j) are created by automated pathway analysis. All pathwaysare involved in keratinocyte differentiation: basal cell regenerationand squamous differentiation.

FIG. 16 A-C: siRNA's against the human R2R¹ and R2R² homologues. Thehuman R2R¹ homologue(s) comprise the FAM25 family encompasing the sevenhuman paralogues designated FAM25A, FAM25B, FAM25C, FAM25D, FAM25E,FAM25G and FAM25HP. These are the human homologues of mouse the murinesequence encoded by the cDNA sequences designated 2200001I15Rik or RIKENcDNA 2200001I15. The similarity of the FAM25 paralogues precludesspecific RTqPCR of the different paralogues. The FAM25 paralogues arealso absent on Affymetrix Human Gene expression arrays such as HTHG-U133 or HT HG-U219. We have selected the RTqPCR primer-probe setHs04194072_m1 (Applied Biosystems) for the measurement of differentialgene expression of the FAM25 family. Applied Biosystems states that thisprimer-probe set doesn't differentiate between the different paralogues.Three siRNA's designed against the FAM25 family were selected on theirability to downregulate Hs04194072_m1 expression in PBEC's. We havenumbered the siRNA's nr 18, 20 and 22 respectively. Graph (a)illustrates the downregulation of FAM25 (Hs04194072_m1) expression (PGK1normalized) after 24 h administration of the respective siRNA's inPBEC's. The human R2R² homologue: Two siRNA's (numbered 15 and 17)designed against C9orf169 (at concentrations of 5 and 20 nMrespectively) were selected on the basis of their ability todownregulate C9orf169 expression in PBEC's. C9orf169 expression wasevaluated by microarray analysis and RTqPCR. Probes for C9orf169 arepresent on the Affymetrix HT HG-U219 Human Gene Expression Array. Graph(b) illustrates the downregulation (assessed by microarray analysis) ofC9orf169 expression after 24 h administration of the respective siRNA'sin PBEC's. The administration of siRNA's directed against the FAM25family or VEGFA has no effect on C9orf169 expression. siRNA's directedagainst R2R¹ (human FAM25 family) and R2R² (human C9orf169) downregulateKRT14 expression and is the response similar to the one observed afteradministration of siRNA's directed against VEGFA and VEGF¹⁶⁵ siRNAmediated knockdown of the FAM25 family and C9orf169 leads todownregulation of KRT14 gene expression (basal cell marker). Thisresponse is most obvious after administration of siRNA's directedagainst C9orf169. Graph (c) illustrates the downregulation KRT14expression (PGK1 normalized) after 24 h administration of the respectivesiRNA's in PBEC's. Included are also the siRNA's directed against VEGFAand KRT14 for reason of comparison.

FIG. 17: is a schematic representation of the effects of the R2Rhomologues. Expression R2R homologues leads to simultaneous modulationof HIF1A signaling (conferring oxygen tolerance) (Box 5), AND modulationof a specific (PERP) anti-apoptotic pathway (Box 4). This will permitthe (re)generation of strong epithelial cells (with a major defensebarrier against stress), without the installment of unlimited growthpotential. In other words, the modulation of a specific anti-apoptoticpathway is not a ‘permit’ for general tolerance to apoptosis. Generaltolerance to apoptosis would lead to the dangerous situation ofimmortalization of the cell: the cell develops into a cancer cell. Thepathway was constructed on the basis of microarray analysis of siRNAmediated knockdown of the human R2R¹ homologue (FAM25 family) and humanR2R² homologue (C9orf169).

FIG. 18: The R²R homologues are essential for ‘Keratinocyte regenerationand differentiation’ (Box 1) and ‘Apoptosis modulation’ (Box 2) in theVEGFA-VEGF¹⁶⁵ pathway. The effects on gene expression by the R2Rhomologues on ‘Keratinocyte regeneration and differentiation’(Box 1) areapparent in FIGS. 18A and 18B. The effects on gene expression by the R2Rhomologues on ‘Apoptosis modulation’(Box 2) are apparent in FIG. 18C.They are the final end-effects of Box 3, 4 and 5.

FIG. 19 A-B: The effect on cellular respiration (see Box 3 in FIG. 17)is highly specific: siRNA mediated knockdown of the R2R homologues willdown-regulate oxidative phosphorylation. Expression of the R2Rhomologues will upregulate oxidative phosphorylation. We can observethis effect at it's most profound in the expression of mitochondrialATP5A1-ATP synthase (H+ transporting) (FIG. 19A). siRNA's directed atthe R2R homologues will downregulate ATP5A1 expression. FIG. 19 Billustrates the expression of ATP5A1 among global changes in geneexpression of the oxidative phosphorylation pathway.

FIG. 20: The effects of the R2R homologues on P53-P63 signaling (see Box4 in FIG. 17). PERP (TP53 apoptosis effector) is downregulated by siRNAmediated knockdown of the human R2R homologues. ‘PERP is a p63-RegulatedGene Essential for Epithelial Integrity. p63 is a master regulator ofstratified epithelial development that is both necessary and sufficientfor specifying this multifaceted program. Perp, a tetraspan membraneprotein originally identified as an apoptosis-associated target of thep53 tumor suppressor, is the first direct target of p63 clearly involvedin mediating this developmental program in vivo’: this was demonstratedby Rebecca A. Ihrie et al. in 2005 (Cell, Vol. 120, 843-856, Mar. 25,2005) (italics quoting author in the abstract of the paper). The graphdemonstrates the downregulation of PERP expression after siRNA mediatedknockdown of the human R2R homologues.

FIG. 21: An overview of the expression of PERP among global changes ingene expression of the P53 pathway after administration of FAM25 nr 20(directed at human homologue R2R¹)

FIG. 22: An overview of the expression of PERP among global changes ingene expression of the P53 pathway after administration of FAM25 nr 18(directed at human homologue R2R¹)

FIG. 23: An overview of the expression of PERP among global changes ingene expression of the P53 pathway after administration of C9orf169 nr15 (directed at human homologue R2R²)

FIG. 24: An overview of the expression of PERP among global changes ingene expression of the P53 pathway after administration of C9orf169 nr17 (directed at human homologue R2R²)

FIG. 25 A-C: The expression of HIF1A (SEE Box 5 IN FIG. 17) is tightlyregulated by the expression of the human R²R homologues. siRNA mediatedknockdown of the human R2R homologues will downregulate HIF1A expression(FIG. 25A). The downstream HIF1A effects on cellular respiration(oxidative phosphorylation) were described in 3 (see FIG. 17 Cellularrespiration). The R2R homologues drive ‘PERP type p53-p63’ and HIF1Aexpression. As such ‘tough’ epithelial cells, equipped with the HIF1Aarmory, could develop unlimited growth potential under the influence ofVEGFA/VEGF¹⁶⁵. However, we observe at the same time that the epithelialcells are precluded from entering an immortal state. Expression of theR2R homologues will act as a brake for the expression of genes thatcould immortalize the cell. This is very obvious in the case of BCL2A1(FIG. 25B), an anti-apoptosis gene whose expression confers resistanceto therapy in cancer cells. siRNA mediated knockdown of the R2Rhomologues leads to upregulation of BCL2A1 expression. The R2Rhomologues act as a brake for BCL2A1 expression. R2R homologues willalso promote the expression of genes that permit cells to enterapoptosis if needed. This is demonstrated by siRNA mediated knockdown ofthe R2R homologues: the knockdown will cause a downregulation MAP2K4(FIG. 25C) a tumor suppressor in lung adenocarcinoma.

DETAILED DESCRIPTION

Table 1. RIKEN cDNA 2200001I15 gene and RIKEN cDNA 2310002J15 gene amongthe top list of genes in SAM analysis of differential gene expression onE16.5 in wild type and Vegf120/120 knockout anti-cytokeratin4-5-6-8-10-13-18 staining cells.

Table 2. RIKEN cDNA 2200001I15 gene and RIKEN cDNA 2310002J15 gene amongthe top list of genes in SAM analysis of differential gene expression onE16.5 in wild type and Vegf120/120 knockout GS-IB4 staining cells.

Table 3. Distribution of embryos according to age and Vegf genotypestatus. wt/wt=homozygous wild type (Vegf+/+), 120/wt=heterozygousVegf120/+, and 120/120=homozygous Vegf120/120 knockout. (NG=notgenotyped because of poor embryologic morphology).

TABLE 1 Probe Set ID Variance UniGene ID Alignments Gene Title GeneSymbol 1448745_s_at 44.327 Mm.1121 chr3: 92166199-9216906

loricrin Lor 1451613_at 25.931 Mm.208047 chr3: 93405151-9341897

hornerin Hrnr 1449986_at 21.731 Mm.160339 chr16: 88647705-886486

RIKEN cDNA 2310034C09 gene 2310034C09Rik 1440186_s_at 19.595 Mm.44242chr5: 36523188-3652342

RIKEN cDNA 2310020A21 gene 2310020A21Rik 1421575_at 18.532 Mm.358728chr16: 88666693-886676

RIKEN cDNA 2310057N15 gene 2310057N15Rik 1453218_at 16.908 Mm.292458chr3: 92764649-9276632

RIKEN cDNA 1110014K05 gene 1110014K05Rik 1459897_a_at 16.677 Mm.250717chr7: 30460230-3046489

suprabasin Sbsn 1420676_at 16.402 Mm.41969 chr3: 92731934-9273371

small proline rich-like 3 Sprrl3 1420358_at 13.819 Mm.353193 chr16:88647705-887093

keratin associated protein 13 Krtap13 1437019_at 13.44 Mm.27156 chr14:33180971-331844

RIKEN cDNA 2200001I15 gene 2200001I15Rik ← 1456248_at 12.776 Mm.46390chr3: 93078529-9307882

RIKEN cDNA 2310002A05 gene ///

2310002A05Rik /// LOC630971 1434227_at 12.373 Mm.268167 chr7:30496664-3049985

keratinocyte differentiation associa

Krtdap 1420677_x_at 12.312 Mm.41969 chr3: 92731934-9273371

small proline rich-like 3 Sprrl3 1439630_x_at 12.212 Mm.250717 chr7:30463567-3046489

suprabasin Sbsn 1420350_at 11.873 Mm.279773 chr3: 92754015-9275571

small proline rich-like 2 Sprrl2 1428781_at 11.64 Mm.30138 chr7:30485165-3048982

RIKEN cDNA 1110014F24 gene 1110014F24Rik 1452732_at 11.02 Mm.183043chr6: 86593812-8659533

RIKEN cDNA 2300003P22 gene 2300003P22Rik 1419394_s_at 10.266 Mm.21567chr3: 90754997-9075596

S100 calcium binding protein A8 (c

S100a8 1453092_at 10.127 Mm.35806 chr3: 93099607-9310107

RIKEN cDNA 2300002G24 gene 2300002G24Rik 1435111_at 9.396 Mm.32861 chr7:24063581-2406386

RIKEN cDNA 2310011E23 gene 2310011E23Rik 1419709_at 9.22 Mm.136573chr16: 36369769-363746

stefin A3 Stfa3 1435761_at 9.202 Mm.383370 chr16: 36196367-362046

stefin A3 Stfa3 1422784_at 7.244 Mm.302399 chr16: 101517949-10152

keratin complex 2, basic, gene 6a Krt2-6a 1435760_at 7.165 Mm.300592chr18: 42299151-422997

cystatin A Csta 1448756_at 6.71 Mm.2128 chr3: 90778558-9078122

S100 calcium binding protein A9 (c

S100a9 1447669_s_at 6.627 Mm.215394 chr13: 13619774-136200

guanine nucleotide binding protein

Gng4 1425336_x_at 5.61 Mm.422886 chr17: 33606481-336107

histocompatibility 2, K1, K region H2-K1 1437145_s_at 5.455 Mm.46431chr2: 25060827-2506109

RIKEN cDNA 2310002J15 gene 2310002J15Rik ← 1420741_x_at 5.247 Mm.291782chr3: 92862612-9286430

RIKEN cDNA 2310069N01 gene 2310089N01Rik 1442339_at 4.062 Mm.187847chr16: 36076009-360811

stefin A2 like 1 Stfa2l1 1427492_at 3.963 Mm.34964 chrX:108755370-108762

premature ovarian failure 1B Pof1b 1418722_at 3.693 Mm.236225 chr9:110265612-110268

neutrophilic granule protein Ngp 1419409_at 3.69 Mm.291769 chr3:92741051-9274165

small proline rich-like 5 Sprrl5 1436936_s_at 3.42 Mm.274770 chrX:99684922-9968596

inactive X specific transcripts Xist 1427262_at 3.408 Mm.274770 chrX:99663093-9968593

inactive X specific transcripts Xist 1430567_at 3.377 Mm.35369 chr18:44114683-441478

serine peptidase inhibitor, Kazal ty

Spink5 1422667_at 3.336 Mm.38498 chr11: 99947848-999520

keratin complex 1, acidic, gene 15 Krt1-15 1448881_at 3.243 Mm.26730chr8: 112464257-112468

haptoglobin HP 1423547_at 2.71 Mm.45436 chr10: 116681442-11668

lysozyme Lyzs 1449586_at 1.939 Mm.4494 chr1: 137687809-137735

plakophilin 1 Pkp1 1449133_at 1.932 Mm.331191 chr3: 92569339-9257128

small proline-rich protein 1A Sprr1a 1449106_at 1.901 Mm.200916 chr11:54746194-547537

glutathione peroxidase 3 Gpx3 1452543_a_at 1.862 Mm.2258 chr19:9150684-9154982 secretoglobin, family 1A, member

Scgb1a1 1450633_at 1.859 Mm.21075 chr13: 3837003-3837919 calmodulin 4Calm4 1459898_at 1.826 Mm.250717 chr7: 30460230-3046489

suprabasin Sbsn 1429565_s_at 1.815 Mm.292457 chr3: 93103210-9310446

late cornified envelope 5A Lce5a 1429540_at 1.727 Mm.34382 chr7:25076381-2507848

cornifelin Cnfn 1453801_at 1.597 Mm.180200 chr3: 94427502-9443259

thioesterase superfamily member 5 Them5 1422672_at 1.568 Mm.140151 chr3:92522208-9252419

small proline-rich protein 1B Sprr1b

indicates data missing or illegible when filed

TABLE 2 Probe Set ID Variance UniGene ID Gene Title Gene Symbol1449586_at 32.877 Mm.4494 plakophilin 1 Pkp1 1423935_x_at 30.173 Mm.6974keratin complex 1, acidic, gene 14 Krt1-14 1460347_at 28.662 Mm.6974keratin complex 1, acidic, gene 14 Krt1-14 1438856_x_at 23.207 Mm.268618serine (or cysteine) peptidase inhibitor, clade B, member 5 Serpinb51422667_at 21.017 Mm.38498 keratin complex 1, acidic, gene 15 Krt1-151422481_at 20.962 Mm.183137 keratin complex 2, basic, gene 1 Krt2-11424096_at 20.14 Mm.383993 keratin complex 2, basic, gene 5 Krt2-51451613_at 20.135 Mm.208047 hornerin Hrnr 1453218_at 18.792 Mm.292458RIKEN cDNA 1110014K05 gene 1110014K05Rik 1441941_x_at 18.5 Mm.268618serine (or cysteine) peptidase inhibitor, clade B, member 5 Serpinb51456203_at 17.757 — RIKEN cDNA 1110020A10 gene 1110020A10Rik 1434227_at16.95 — keratinocyte differentiation associated protein Krtdap1429067_at 16.396 — calpain, small subunit 2 Capns2 1440186_s_at 16.186Mm.378865 Transcribed locus — 1419709_at 15.648 Mm.136573 stefin A3Stfa3 1422939_at 14.807 Mm.337362 serine (or cysteine) peptidaseinhibitor, clade B (ovalbumin), member 3B Serpinb3b 1422308_a_at 14.219Mm.20973 lectin, galactose binding, soluble 7 Lgals7 1437019_at 13.876Mm.27156 RIKEN cDNA 2200001I15 gene 2200001I15Rik ← 1450633_at 13.842Mm.21075 calmodulin 4 Calm4 1421117_at 12.773 Mm.336625 dystonin Dst1421752_a_at 12.718 Mm.268618 serine (or cysteine) peptidase inhibitor,clade B, member 5 Serpinb5 1418799_a_at 12.316 Mm.1225 procollagen, typeXVII, alpha 1 Col17a1 1453801_at 11.671 Mm.180200 thioesterasesuperfamily member 5 Them5 1422940_x_at 11.333 Mm.337362 serine (orcysteine) peptidase inhibitor, clade B (ovalbumin), member 3B Serpinb3b1436392_s_at 11.112 Mm.3629 transcription factor AP-2, gamma Tcfap2c1452166_a_at 10.901 Mm.22662 keratin complex 1, acidic, gene 10 Krt1-101449938_at 10.491 Mm.1001 placental protein 11 related Pp11r1421040_a_at 10.27 Mm.371562 glutathione S-transferase, alpha 2 (Yc2)Gsta2 1437232_at 10.198 Mm.107214 bactericidal/permeability-increasingprotein-like 2 Bpil2 1424623_at 9.782 Mm.268618 serine (or cysteine)peptidase inhibitor, clade B, member 5 Serpinb5 1418748_at 9.085Mm.20940 caspase 14 Casp14 1418158_at 9.029 Mm.20894 transformationrelated protein 63 Trp63 1430551_s_at 8.917 Mm.195937 lipase-like,ab-hydrolase domain containing 3 LipI3 1430550_at 8.679 Mm.195937lipase-like, ab-hydrolase domain containing 3 LipI3 1435761_at 8.652Mm.136573 stefin A3 Stfa3 1448397_at 8.545 Mm.25652 gap junctionmembrane channel protein beta 6 Gjb6 1459898_at 8.407 Mm.250717suprabasin MGI: 2446326 1422672_at 8.373 Mm.140151 small proline-richprotein 1B Sprr1b 1459897_a_at 8.371 Mm.250717 suprabasin MGI: 24463261428781_at 8.218 Mm.30138 RIKEN cDNA 1110014F24 gene 1110014F24Rik1419492_s_at 8.128 Mm.5341 defensin beta 1 Defb1 1439183_at 8.102Mm.218784 N-acylsphingosine amidohydrolase (alkaline ceramidase) 3 Asah31419491_at 8.028 Mm.5341 defensin beta 1 Defb1 1427263_at 7.99 —inactive X specific transcripts Xist 1416930_at 7.471 Mm.878 lymphocyteantigen 6 complex, locus D Ly6d 1435760_at 7.251 Mm.300592 cystatin A/// similar to Stefin homolog MGI: 3524930 /// LOC547252 1439630_x_at7.031 Mm.250717 suprabasin MGI: 2446326 1435639_at 6.509 — RIKEN cDNA2610528A11 gene 2610528A11Rik 1424976_at 6.23 Mm.120274 ras homolog genefamily, member V Rhov 1455519_at 6.163 Mm.383274 desmoglein 1 beta Dsg1b1455715_at 5.932 Mm.373656 PREDICTED: Mus musculus RIKEN cDNA 2700099C18gene — (2700099C18Rik), mRNA 1434534_at 5.693 — — — 1426048_s_at 5.603Mm.85544 transcription factor AP-2, alpha Tcfap2a 1449500_at 5.535Mm.66015 serine (or cysteine) peptidase inhibitor, clade B, member 7Serpinb7 1430582_at 5.508 Mm.133101 SNF2 histone linker PHD RINGhelicase Shprh 1435670_at 5.464 Mm.137021 transcription factor AP-2 betaTcfap2b 1422588_at 5.427 Mm.358617 keratin complex 2, basic, gene 6bKrt2-6b 1445187_at 5.408 Mm.329504 RIKEN cDNA 9430070O13 gene /// genemodel 979, (NCBI) 9430070O13Rik /// Gm979 1421996_at 5.28 Mm.85544transcription factor AP-2, alpha Tcfap2a 1423323_at 5.278 Mm.154045tumor-associated calcium signal transducer 2 Tacstd2 1452228_at 5.242Mm.257819 RIKEN cDNA 4930451A13 gene 4930451A13Rik 1449959_x_at 5.214Mm.23784 small proline rich-like 9 Sprrl9 1419731_at 5.211 Mm.14098cytochrome P450, family 2, subfamily b, polypeptide 19 Cyp2b191442279_at 5.14 Mm.312133 Enhancer of polycomb homolog 1 (Drosophila)(Epc1), transcript variant 1, Epc1 mRNA 1455408_at 5.008 Mm.24880 RIKENcDNA 4732472I07 gene 4732472I07Rik 1416271_at 4.925 Mm.28209 PERP, TP53apoptosis effector Perp 1418722_at 4.888 Mm.236225 neutrophilic granuleprotein Ngp 1437351_at 4.809 Mm.224814 CXXC finger 4 Cxxc4 1440162_x_at4.662 Mm.208144 hypothetical protein A630043P06 A630043P06 1426641_at4.647 Mm.266679 tribbles homolog 2 (Drosophila) Trib2 1442349_at 4.643Mm.259334 RIKEN cDNA C630028N24 gene C630028N24Rik 1425624_at 4.593Mm.209005 EPM2A (laforin) interacting protein 1 Epm2aip1 1460038_at4.577 Mm.297371 POU domain, class 3, transcription factor 1 Pou3f11440523_at 4.562 Mm.98096 retinal short chain dehydrogenase reductase 2MGI: 2668443 1431211_s_at 4.438 Mm.180200 thioesterase superfamilymember 5 Them5 1447329_at 4.351 — — — 1443687_x_at 4.344 — — —1420988_at 4.306 Mm.311585 polymerase (DNA directed), eta (RAD 30related) Polh 1456248_at 4.189 Mm.46390 RIKEN cDNA 2310002A05 gene2310002A05Rik 1430000_at 4.157 — RIKEN cDNA B230117O15 geneB230117O15Rik 1446490_at 4.154 Mm.29966 Polypyrimidine tract bindingprotein 2, mRNA (cDNA clone MGC: 11671 Ptbp2 IMAGE: 3709255)1441909_s_at 4.1 Mm.225253 RIKEN cDNA 9530066K23 gene 9530066K23Rik1427747_a_at 4.035 Mm.9537 lipocalin 2 Lcn2 1437145_s_at 3.994 Mm.46431RIKEN cDNA 2310002J15 gene 2310002J15Rik ← 1419463_at 3.985 Mm.20897chloride channel calcium activated 2 Clca2 1441440_at 3.976 Mm.277366autophagy-related 4C (yeast) Atg4c 1437705_at 3.854 — — — 1418028_at3.846 Mm.19987 dopachrome tautomerase Dct 1442786_s_at 3.804 Mm.270469DNA segment, Chr 5, Brigham & Women's Genetics 0860 expressed D5Bwg0860e

TABLE 3 E7733 E12.5 A wt/wt B wt/120 C 120/120 D wt/120 E wt/120 F wt/wtG wt/wt H 120/120 E7734 E12.5 A 120/120 B wt/120 C wt/120 D wt/120 Ewt/120 F wt/120 G wt/120 H 120/120 I wt/wt K 120/120 E7232 E14.5 A120/wt B 120/wt C 120/120 D wt/wt E 120/wt F 120/wt G wt/wt H 120/120 I120/wt K wt/wt L wt/wt E7494 E14.5 A 120/120 B wt/wt C 120/wt D 120/wt E120/120 F 120/wt G 120/wt H 120/wt I wt/wt E7119 E16.5 A 120/wt B 120/wtC 120/120 D 120/wt E 120/wt F 120/wt G 120/120 H 120/wt I wt/wt J 120/wtK 120/wt L 120/wt E7477 E16.5 A 120/120 B wt/wt C ? D 120/+ E wt/wt Fwt/wt G ? H 120/wt I 120/wt K 120/wt L 120/120 E7478 E16.5 A 120/120 Bwt/wt C 120/wt D 120/wt E 120/wt F 120/120 G wt/wt H 120/wt I 120/wt Kwt/wt L 120/120 M wt/wt

Introduction

The R2R genes, transcripts and respective proteins were discovered in aVEGF mouse knockout model. The Vegf120/120 knockout mouse is unable toproduce the Vegf¹⁶⁴ and Vegf¹⁸⁸ isoforms (Vegf¹⁶⁴ is the homologue ofhuman VEGF¹⁶⁵). The lungs of Vegf120/120 knockout mice are hypoplasticat birth and peripheral airway and vascular differentiation becomesseverely impaired in Vegf120/120 knockout embryonic lungs. A genomicsapproach led to the discovery that Vegf¹⁶⁴ in the mouse and VEGF¹⁶⁵ inman drive a very specific gene expression program. This program consistsof two components. The first component is the (re)generation of basalcells of the airway epithelium: basal cells are the source of at leastthe cellular population of the proximal airways. The basal cells alsogenerate strong intercellullar connections by building hemidesmosome andfocal adhesion connections. The basal cells strengthen theirintracellular architecture by the intermediate filament proteins KRT14and KRT5. These two proteins are glued to the (hemi)desmosome.

The second component is a differentiation program: it's a ‘fortificationprogram’ (‘squamous differentiation’) of the cells lining the airways.These cells need to be tough at birth, as they will be exposed tomechanical stress and high levels of oxygen. The fortification is madepossible by a family of proteins that strengthen cellular architecture.This family of proteins consists of the intermediate filament group andthe proteins that strengthen the intermediate filaments (SPRR proteins,LOR, HRNR, etc). These two programs can be summarized under the headings‘keratinocyte differentiation’, ‘epidermal cell differentiation’,‘intermediate filament reorganization’, ‘cornified envelope’,‘keratinocyte differentiation’. ‘cytoskeleton remodeling keratinfilaments’.

While it is tempting to use the VEGF¹⁶⁵ protein for the regeneration ofdamaged lungs in humans, VEGFA and VEGF¹⁶⁵ are important regulators of avast diversity of processes in the organism. Hence, the administrationof VEGFA or VEGF¹⁶⁵ would result in too many side effects.

Two novel genes (R2R¹ and R2R²) were discovered in the gene expressionprogram that's driven by Vegf¹⁶⁴ in the mouse and VEGF¹⁶⁵ in man. Thesenovel genes, their transcripts and translated proteins are not relatedto the genes and their downstream products of the basal and squamousgene expression program.

Experiments were undertaken to demonstrate that these genes areimportant modulators of the basal and squamous differentiation program.One of the most important findings of these experiments is that thesegenes are important modulators of HIF1α and PERP expression in the cell.In our experiments, the R2R genes are positive regulators andinterference with this mechanism opens therapeutic possibilities incancer therapy.

Material and Methods

Mouse Embryos and Tissue Processing.

All animal experiments were approved by the Animal Ethics Committee ofLeiden University Medical Center and performed according to the Guidefor the Care and Use of Laboratory Animals published by the NIH.Heterozygous Vegf+/120 mice were crossed to obtain Vegf120/120 embryosand Vegf+/+ wild type littermates. The morning of the vaginal plug wasdefined embryonic day (E) 0.5. Pregnant females were sacrificed bycervical dislocation. E12.5, 14.5 and 16.5 embryos were isolated insterile PBS. The embryonic thoraces were carefully dissected inRnase-free conditions, placed in tissue freezing medium (TBS, TriangleBiomedical Sciences, Durham N.C.), frozen and stored at −80° C. Thedistribution of embryos according to age and maternal origin isrepresented in Table S1.

Cryostat sections (8 μm) were cut and attached to SuperFrost Plusmicroscope slides (Menzel Gmbh & Co KG, Braunschweig Germany).Sectioning and further immunohistochemical processing of embryonicthoraces of different ages was performed at random.

Immunohistochemistry and Laser Capture Microdissection.

Three tissue sections from each embryonic thorax were selected at thelevel of the biventricular view of the heart. These wereimmunohistochemically processed in one batch. The cryostat sections werefixed by placing the slides in cold acetone (4° C.) during 2 minutesafter removal from the −80° C. freezer. All further immunohistochemicalsteps were performed at 4° C., and all buffers and antibody solutionskept at 4° C. Rnase free PBS or D-PBS buffer was prepared by dilutingRNAsecure (25×, AM7006, Ambion TX) to 1× in the desired buffer. Allantibody solutions were prepared in PBS whereas the isolectin GS-IB₄conjugate was diluted in D-PBS. Superase.In (AM2696, Ambion, Austin,Tex.) was added to each antibody solution in a final concentration of1U/μl. The slides were air-dried and the tissue sections circumscribedwith a hydrophobic barrier pen. After placing the slides on a cold metalblock (4° C.), 30 ml of PBS was applied to each tissue section anddrained off. Subsequently, 30 ml of mouse anti-keratin pan (4, 5, 6, 8,10, 13, 18) monoclonal antibody (MAB1636, Chemicon) in a concentrationof 10 μg/100 μl was dripped on the specimen. The antibody solution wasdrained off after 2 minutes and the tissue section gently rinsed with250 ml of PBS. Thirty μl of Alexa-fluor-488 chicken anti-mouse IgG (H+L)conjugate (A21200, Invitrogen CA) in a concentration of 10 μg/100 μl wasthen applied for 2 minutes, followed again by a gentle wash with 250 μlof PBS. Finally, a third cycle of 2 minutes staining with 30 μl ofisolectin GS-IB₄ Alexa Fluor 594 conjugate (I21413, Invitrogen, CA) in aconcentration of 10 μg/100 μl completed the staining procedure. Thetissue sections were dehydrated at room temperature: 75% EtOH (30 sec),95% EtOH (30 sec), 100% EtOH (30 sec), 100% EtOH (120 sec), xylene (180sec). Laser capture microdissection was performed on a VeritasMicrodissection Instrument (Arcturus Bioscience Inc., Mountain View,Calif.) immediately after the dehydration steps. We dissected 3×300 to400 cells (as triplicate samples) from intrapulmonary airways or bloodvessels in the embryonic lungs of three tissue sections at the level ofthe biventricular view of the heart. Cells staining for mouseanti-keratin pan monoclonal antibody/chicken anti-mouse IgGAlexa-fluor-488 conjugate were identified as green fluorescent cells(blue filter). These green fluorescent cells were defined as airwayepithelial cells (ker+ cells) and were randomly dissected, irrespectiveof their proximal or distal airway morphology. Cells staining forisolectin GS-IB₄ Alexa-fluor-594 conjugate were identified as redfluorescent cells (green filter). These cells were defined asmesenchymal cells with endothelial features (il+ cells). Staining ofcells for both markers was not observed on the three embryonic timepoints (E 12.5, 14.5, 16.5). In fact, the green and red fluorescentcells could be observed as a positive/negative image from each other.The microdissected ker+ or il+ cells were collected in a Gene Amp tube(Applied Biosystems, Foster City Calif.) filled with 75 μl of RNeasylysis buffer (RLT; Qiagen, Hilden, Germany) containing 0.14 Mβ-mercaptoethanol and 200 ng polyinosinic acid (Sigma).

RNA Isolation, Amplification, Labeling, and Microarray Hybridization.

Laser-captured samples were incubated at 42° C. for 20 minutes and thenchilled on ice. Samples were stored at −80° C. until further processing.After thawing, an equal volume of 70% ethanol was added to each sampleand then transferred to RNeasy MinElute Spin Columns (Qiagen). RNA wascleaned up according to the manufacturer's instructions, eluted in 14 μlof RNase-free water, and adjusted to 4 μl by vacuum drying. Two roundsof linear mRNA amplification were needed to generate sufficientquantities of cRNA. Two-cycle cDNA synthesis and synthesis ofbiotin-labeled cRNA was performed according to the GeneChip EukaryoticSample and Array Processing Manual (Affymetrix, Santa Clara, Calif.). As“spike-in” controls, the GeneChip Poly-A RNA control kit (Affymetrix)was used. MEGAscript T7 kit (Ambion, Austin, Tex.) was used for in vitrotranscription of the second cDNA strand in the first round ofamplification, yielding 112 to 457 ng of aRNA. The second round ofamplification, starting from 100 ng of first round aRNA, yielded 11 to86 μg of cRNA using the GeneChip in vitro transcription (IVT) labellingkit. Labeled RNA was hybridized to mouse genome MG-430_(—)2.0 GeneChiparrays (Affymetrix). Hybridization was performed using 12.5 μg ofbiotin-labeled RNA at 45° C. for 16 hours under continuous rotation.Arrays were stained in Affymetrix Fluidics stations usingstreptavidin-phycoerythrin (SAPE), followed by staining withanti-streptavidin antibody and a second SAPE staining. Subsequently,arrays were scanned with an Agilent Laserscanner (Affymetrix).

Statistical Analysis.

The Affymetrix probe level data were summarized using FARMS (FactorAnalysis for Robust Microarray Summarization)¹. Raw intensities werelog₂ transformed to get data normally distributed. First, anunsupervised multivariate projection method, Spectral Map Analysis², wasapplied to reduce the complexity of highly dimensional data (n genes vs.p samples). Spectral Map Analysis provides an unbiased identification ofthe predominant clusters of genes and subjects that are present in thedata set. Second, tests for differential gene expression between the twocellular origins (ker+ versus il+ cells) was performed in LIMMA (LinearModels for Microarray Data)³, as this method uses information acrossgenes making the analyses stable even for experiments with small numberof arrays³. Third, differences between Vegf120/120 knockout and wildtype littermates in expression profiles over embryonic age were testedthrough a two-way interaction of Vegf genotype and time, again usingLIMMA³. The data of E12.5 and E14.5 were pooled, as we were onlyinterested in the contrasting time profile of E16.5 versus E14.5 andE12.5. This test was performed on the ker+ and il+ samples separately,because these two tissues originated from the same embryos. Models likeLIMMA assume that all the samples have been randomly and independentlycollected. Correction for this dependency would have required toocomplex models if the ker+ and il+ would have been analysedconcurrently. Genomic variation from the single interaction of tissuetype (ker+ versus il+ samples) was also uncovered by LIMMA analysis.Allocation of differential expression along the whole genome wasperformed using MACT (Microarray Chromosome Analysis Tool).

Results/Discussion

At birth, O₂ and CO₂ need to be exchanged in the lung across a largeinterface of distal airways and blood vessels. Embryonic lungdevelopment in the mouse undergoes a striking shift at E (=postconceptual day) 16.5¹. At this time, the intertwined airway and vasculartrees sprout by multiplying and refining their distal branches. Thedistal airways or respiratory tubules multiply by subdivision into thinwalled sacculi before birth. These sacculi eventually develop intopostnatal alveoli². Thin walled airways require flat cells to facilitategas transport. The phenotypic differentiation to flat airway cells,which occurs around E16.5, is therefore a crucial phase in embryoniclung development. Epithelial cells covering the airways originate frombranching foregut endoderm. From E16.5, epithelial cells in the distalairway start to flatten out whereas proximal cell preserve theircolumnar shape. The most distal of these cells will line the sacculi andalveoli and develop a flat or even squamous morphology by E18.5. Thecapillaries are lined with flat endothelial cells and represent thedistal blood vessels of the vascular tree. Endothelial cells coveringpulmonary blood vessels derive from mesodermal mesenchyme. Their growthneeds to closely match the growth of their epithelial counterparts inorder to provide the large alveolar-capillary interface over which gasexchange initiates at birth. Reciprocal crosstalk between endodermallyderived airway epithelium and surrounding mesodermal mesenchymeinitiates at early lung morphogenesis^(3,4). Starting at E9.5, Fgf10produced by mesenchymal cells in surrounding mesoderm is the mostimportant cue for branching endoderm. Close interaction with at leastShh, Bmp, TGF-β, and Wnt signaling factors modulates this earlybranching mechanism. However, the molecular machinery underpinning thelater cellular phenotypical changes and epithelial-endothelial crosstalkat E16.5 is less understood.

In order to gain further insight into late lung differentiation afterE12.5, we developed a RNA-friendly immunohistochemical staining protocolfor laser capture microdissection of epithelial cells in the developingairway. We reasoned that downstream gene expression profiling of RNAisolated from airway cells sharing a common epithelial antigen atdifferent embryonic ages, should highlight their transcriptional changesover time. This program should at least reflect epithelial features,preferably of the pulmonary airway type. The same assumption was testedon pulmonary cells marked for an endothelial marker universallyexpressed at different embryonic ages. Additionally, a mouse knockoutmodel with late abnormal pulmonary branching morphogenesis wasincorporated in this approach. We chose the Vegf120/120 model, asperipheral airway and vascular differentiation^(5,6,7) becomes severelyimpaired in these Vegf120/120 knockout embryonic lungs. Wild typeepithelial and endothelial cells were expected to express a set ofairway and vascular differentiation genes, lacking in their Vegf120/120knockout counterparts. Vegf120/120 mice lack VEGF-A isoforms 164 and188, but still express isoform 120. VEGF-A isoforms 164 and 188 (VEGF164and VEGF188) are more tightly bound to the extracellular matrix than themore soluble VEGF120 variant, and concentrate locally around distalairways. The standard view states that pulmonary epithelial cellssecrete these VEGF-A isoforms, whereby VEGF164 and VEGF188 encouragelocal growth of pulmonary endothelial cells through stimulation ofreceptor tyrosine kinases Flk1 (VEGF receptor-2) and Flt1 (VEGFreceptor-1). Confined expansion of endothelial cells refines thepulmonary vascular tree and allows a matching growth of epithelialcells^(8,9). This epithelial-endothelial crosstalk permits gas exchangeat birth by the formation of a close bond between the alveoli of thedistal airways and the capillaries of the lung vasculature. However,this type of interaction cannot explain the presence of VEGF-A inmesenchymal cells surrounding the distal airway epithelial cells.

Immunohistochemical staining was performed on frozen tissue sections cutfrom embryonic thoraces isolated at E12.5, E14.5 and E16.5 (FIG. 2). Thegenomic distribution of the embryos is shown in Table S1. We selectedantibodies exhibiting sufficient bandwidth for binding epithelial orendothelial antigens in the embryonic time frame of our study.Anti-cytokeratin (directed against cytokeratin 4, 5, 6, 8, 10, 13, and18) was chosen for labelling of epithelial cells (ker+ cells) lining theairways, as primitive and differentiated epithelial cells globallyexpress intermediate filaments of different keratins. Endothelial cellsin the same tissue section were stained with isolectin GS-IB₄ (Griffoniasimplicifolia) Alexa-fluor-594 conjugate, which binds early¹⁰ and lateendothelial cells^(11,12) in the mouse (il+ cells). Staining of cellsfor both immunohistochemical markers was not observed on the threeembryonic time points. Three hundred to four hundred ker+ and il+ cellswere selectively isolated by laser capture microdissection. Two roundsof linear mRNA amplification yielded sufficient cRNA for hybridizationto Affymetrix Mouse 430_(—)2.0 Genechips.

First, we examined if downstream gene expression profiling wasreflecting differentiation with respect to embryonic age and epithelialversus endothelial origin. Exploratory, unsupervised analysis of geneexpression data revealed that gene expression changes during embryonicdevelopment accounted for the largest variation (35%) in the data set.This variation was graphically well represented in the first principalcomponent (X-axis or PC₁) of a spectral map¹³ (FIG. 3). Genes displayingthe strongest expression changes over the three embryonic stages lie atthe extremes of the X-axis. One of these genes, surfactant associatedprotein C (Sftpc), is known to have an important physiologicalupregulation during embryogenesis, and sufficient amounts of its proteinproduct are necessary for normal breathing at birth^(14,15). The secondprincipal component (Y-axis or PC₂), explaining another 17% of thevariation in the dataset, could be assigned to gene expressiondifferences in cellular origin. Some of the most extreme probe sets onthe PC₂ axis represent genes that are known to be highly characteristicfor either pulmonary epithelium or endothelium. Among the eight mostextreme probe sets, we identified CD 93 antigen (CD93)¹⁶ and claudin 5(Cldn5)¹⁷ as illustrative endothelial genes, and on the opposite side ofthe Y-axis, forkhead box A1 (Foxa1)¹⁸ and keratin 8 (Krt8)¹⁹ asexpressive epithelial genes. Superposition of the different samples onthe spectral map showed their distribution along the first two principalcomponents. The ker+ and il+ groups were clearly separated according totheir cellular origin along PC₂ (FIG. 3). Ker+ samples grouped towardsepithelial type genes and il+ samples assembled towards endothelial typegenes. Both cellular origins gathered together on PC₁ at the sameembryonic ages. This indicates that the overall developmental geneexpression changes are similar for epithelial (ker+) and endothelial(il+) cells. As a whole, the samples cluster together with respect tocellular origin (ker+ versus il+ cells) and embryonic age when applyingan unsupervised data-driven analysis. The spectral map analysisunderscores that selective laser capture microdissection revealed a goodresolution of gene expression profiles. An independent supervisedunivariate (gene-by-gene) analysis of the effect of tissue origin of thesamples (ker+ versus il+) further confirmed that ker+ and il+ cellscorresponded at the genomic level with epithelial or endothelial cells,respectively (FIG. 1).

Next, the transcriptional profile associated with abnormal branchingmorphogenesis in the Vegf120/120 knockout phenotype was charted in il+and ker+ cells. For every gene, we tested whether its expression profileover embryonic age differed significantly between Vegf120/120 knockoutand wild type (Vegf+/+) littermates. This difference in age dependantexpression profile between Vegf+/+ and Vegf120/120 unfolded a genomicroadmap in three directions. There were several genes identified withonly in the Vegf+/+ genotype a clear upregulation over embryonic age,e.g. Hmr (FIG. 2). The Vegf120/120 genotype showed an impairment in thatage dependant induction.

First, the cause of the structural deficit in the airways of Vegf120/120knockout lungs became apparent. Wild type ker+ cells highly expressed asurplus of 44 epithelial-specific genes on E16.5 compared to theirVegf120/120 knockout counterparts. A group of genes of the EpidermalDifferentiation Complex (EDC) dominated the expression profile (FIG. 4).S100a8 and S100a9 figured among this EDC subset and are known to beVEGF-A responsive²⁰ chemoattractants. Other elements of the EDC such assmall proline rich region (Spry) genes and late constituents of theepidermal cornified envelope were also present. Cytoskeletal keratinKrt2-6 was co-expressed with this EDC subset in wild type ker+ cells onE16.5. Serine-cysteine proteinase inhibitors and the three genes of theSCC (stratified epithelium-secreted protein gene) complex completed thecohort of upregulated genes (FIG. 4).

Study of the interaction between embryonic age and genotype in wild typeil+ cells uncovered again a profound upregulation of a limited set ofgenes on E16.5. This response led towards adoption of anepithelial-specific transformation program in wild type il+ cells onE16.5 compared to Vegf120/120 knockout cells. As in wild type ker+cells, the EDC cluster, the SCC cluster, the cysteine proteinaseinhibitors and exclusive keratin genes were clearly upregulated overembryonic age. Riken1110020A10, corresponding to the Dsc1 gene, washighly upregulated in wild type il+ cells on E16.5. Furthermore, Pkp1(plakophilin 1) displayed an identical transcriptional profile on E16.5(FIG. 5). A highly logical pattern appeared to drive the clusteredupregulation of these genes. Keratins are intermediate filament proteinslending structural strength to the cell, most typically in epithelialcells²¹. Proteins encoded by the EDC and SCC cluster, andserine-cysteine proteinase inhibitors fortify this keratin network. Dsc1(desmocollin 1) codes for one of the proteins shaping thedesmosomes^(22,23). Intermediate keratin filaments are linked tointercellular desmosomes that form the cell junction together with gapand adherens junctions. The protein encoded by Pkp1 is most of all apositive regulator of desmosomal protein content^(24,25) and is aconstituent of the desmosomal complex itself. Pkp1 also linksintermediate keratin filaments to the cadherin proteins of the adherensjunctions. These results identify receptor tyrosine kinase stimulationby VEGF-A isoforms 164 and 188 as a master switch in the assembly of thedesmosomal/intermediate filament machinery in the lung. This mechanismadds another building block to the cytoskeletal and intercellulararchitecture on top of the Wnt/β-catenin-dependent adherens junction(E-cadherin). In fact, the upregulation of Eps811 on E16.5 in wild typeil+ cells revealed even direct interference with actin, a key structuralprotein unrelated to the intermediate filament system. The coordinatedand clustered expression of these cytoskeletal and desmosomal genespermits shaping of flat or squamous cellular arrangements in distalairways.

Second, aside from activation of genes coding for specific structuralproteins, an intriguing finding was the upregulation of Mapkapk3 in wildtype il+ cells on E16.5. Mapkapk3 integrates ERK and p38 signaling²⁶pathways in stress and mitogen responses such as VEGF-A stimulation ofthe endothelial cell. Cdkn2b (p15^(ink4b) or Ink4b), part of theInk4b-ARF-Ink4a tumor suppressor locus, was simultaneously upregulated.Substantial evidence points to suppression of this locus by associatedpolycomb group (Pcg) repressor complexes. Derepression of the locusoccurs upon dissociation of Peg complexes by activation oroverexpression of Mapkapk3²⁷. This brake pedal in the cell cycle allowsdifferentiation during proliferative stimuli²⁸ and was demonstrated herefor the first time in vivo. In effect, cell cycle arrest permittingepithelial transformation of il+ cells is VEGF164- and VEGF188-dependentin the lung. Intriguingly, upregulation of Krt5, Krt14 and Tcfap2c wasstrikingly similar to the expression fingerprint of the basal cellprogenitor in airway epithelium²⁹. The basal cell phenotype only appearsat birth in the pulmonary airway epithelium and typically bindsisolectin. On the other hand, Krt1, EDC and SCC cluster gene expressionis a squamous differentiation program. The protein ANp63 and TAap63drive the keratin progenitor and squamous differentiation programrespectively. The Trp63 gene coding for these two proteins wasupregulated in the wild type il+ cells. As mentioned, staining of cellsfor both anti-cytokeratin (anti-4, 5, 6, 8, 10, 13, and 18) andisolectin GS-IB₄ was not observed at the time points studied. The lackof Krt1 and Krt14 binding by the anti-cytokeratin antibody, allowedtherefore the identification of the specific epithelial transformationprogram of wild type il+ cells at E16.5. It seems unlikely that ker+epithelial cells generate this epithelial transcriptional program. Thiswould require losing the binding capacity for the anti-cytokeratinantibody in order to escape ker+ labeling. At the same time, the ker+cells would have to acquire exclusive isolectin GS-IB4 staining. As aresult, we propose that pulmonary il+ cells harbor a reservoir of cellsgrowing into epithelial maturity at E16.5. In other words, pulmonarymesenchymal il+ cells encompass cells with endothelial and epithelialcompetence.

Third, the gene represented by Affymetrix probe 1437019_at (RIKEN cDNA2200001I15 gene) and the gene represented by Affymietrix probe1437145_s_at (RIKEN cDNA 2310002J15 gene) were both upregulated on E16.5in wild type anti-cytokeratin 4-5-6-8-10-13-18 staining epithelial cellsand wild type GS-IB4 binding cells. We found these two genes (lackingbiological annotation) to be tightly co-expressed with the squamous andbasal cell transcriptional program. They play an important role inproduction and regeneration of differentiated airway cells. (FIG. 6,FIG. 7, Table 1, Table 2). Starting from the longest contig constructedfrom sequenced clones, we discovered the human homologue of the RIKENcDNA 2200001I15 gene transcript (FIG. 8) and RIKEN cDNA 2310002J15 genetranscript (FIG. 10). Furthermore, protein sequence alignments confirmedthe existence of a human homologous protein for the respectivetranslated protein of the RIKEN cDNA 2200001I15 gene (FIG. 9) and RIKENcDNA 2310002J15 gene (FIG. 11). We suggest the name R2R¹ for themammalian homologues of the RIKEN cDNA 2200001I15 gene, transcript andprotein, and R2R² for RIKEN cDNA 2310002J15 gene, transcript and proteinas these genes and their protein products are responsible forregenerative function in the respiratory system.

Fourth, we found that VEGF-A is expressed in ker+ and il+ cells,independent of wild type or Vegf120/120 knockout status. Moreover, thegene coding for VEGF receptor 1 (Flk-1 or Kdr) was abundantly expressedin il+ cells, but also increased significantly as early as E14.5 in ker+cells. This pattern of VEGF-A and VEGF receptor expression challengesthe classic viewpoint of mesenchyme passively waiting for a VEGF-Astimulus from pulmonary epithelium. It coincides essentially with recentwork demonstrating the need of endogenous VEGF-A expression andautocrine signaling in survival of endothelial cells^(30,31). From thegenomic footprint of wild type versus Vegf120/120 knockout il+ cells, itbecame also apparent that the il+ cells are the ones responsible forsending epithelial transformation stimuli. Upregulation of Fgfbp1,Lgals7, Lgals3 and Il18 in wild type il+ cells on E16.5 representedthese stimuli. The upregulation of the gene encoding fibroblast growthfactor-binding protein 1 (Fgfbp1) indicates that primordialFGF-controlled lung budding is also at work in the final stages of lungdifferentiation. Fgfbp1 acts by concentrating FGF2 and is a finemodulator of growth and differentiation of epithelial tissues inresponse to FGF stimuli from mesenchyme. The FGF receptor 2 gene (Fgfr2)was in this respect richly expressed in the ker+ and il+ compartment.

In summary, selective laser capture microdissection of cells sharing aspecific marker at different embryonic ages was performed. This alloweddifferentiation of gene expression profiles with respect to embryonicage, cellular origin and Vegf genotype. The transcriptional programrevealed by this approach highlighted the importance of the intermediatefilament and desmosomal network in the refinement of pulmonaryarchitecture. This mechanism adds another building block to thecytoskeletal and intercellular architecture on top of theWnt/β-catenin-dependent adherens junction (E-cadherin). Intermediatefilaments provide the necessary strength to cells that will be exposedto vast amounts of mechanical and oxidant stress, Not surprisingly,pulmonary cells adopt the same defensive genomic program as skin cells,which are exposed to the same challenges. In parallel with cytoskeletalsophistication, the basal cell progenitor program is acquired by i/+cells late in embryonic life, and is VEGF 164-188-dependent,

We have demonstrated that downregulation of FAM25 family anddownregulation of C9orf169 gene expression lowers KRT14 expression.Further, we know that expression of the FAM25 family (the human R2R¹homologue) and C9orf169 (the human R2R² homologue) is upregulated byVEGFA/VEGF¹⁶⁵. How do these genes act in the VEGFA/VEGF¹⁶⁵ pathway?siRNA mediated knockdown of the R2R homologues can be used to uncoverthe specific role of the R2R homologues in the pathway, Indeed the R2Rhomologues' role is to modulate the VEGFA/VEGF¹⁶⁵ response in the cell.The basal expression of VEGFA and the VEGF¹⁶⁵ isoform is high in thepulmonary epithelium. Epithelial functions and structures are far morecomplex than their endothelial counterparts. Therefore, the typicalVEGFA/VEGF¹⁶⁵ effects of ‘grow and multiply’ in the endothelium need tobe more refined in the endothelium. How is this realized? Expression R2Rhomologues leads to simultaneous modulation of HIF1A signaling(conferring oxygen tolerance), AND modulation of a specific (PERP)anti-apoptotic pathway. This will permit the (re)generation of strongepithelial cells (with a major defense barrier against stress), withoutthe installment of unlimited growth potential. In other words, themodulation of a specific anti-apoptotic pathway is not a ‘permit’ forgeneral tolerance to apoptosis. General tolerance to apoptosis wouldlead to the dangerous situation of immortalization of the cell: the celldevelops into a cancer cell.

This study sheds new light on certain human lung diseases. In pretermneonates, late embryonic pulmonary development is disrupted when theinfant is prematurely delivered. Insufficient levels of surfactantprotein in pulmonary alveoli lead to severe respiratory distress in alarge group of premature infants. Instillation of surfactant in theneonatal lung has aided the prevention and treatment of respiratoryfailure in premature infants. However, high oxygen levels and tensilestress-forces of mechanical ventilation still lead to chronic lungdamage or bronchopulmonary dysplasia (BPD). Accelerating squamousdifferentiation in distal airways of premature infants may prevent thisdebilitating condition. The intermediate filament network andsynchronized replenishment of the basal cell pool may also be ofparamount importance in the search for a cure of certain adult lungdiseases. Adoption of a squamous phenotype with expression of some EDCcluster genes characterizes squamous metaplasia in the airways of adultswith chronic obstructive pulmonary disease (COPD). This defensemechanism against noxious stimuli is however accompanied with loss ofregenerative basal cells³². In contrast, the embryo succeeds indeveloping a squamous differentiation program while simultaneouslybuilding a basal cell reservoir by a well-defined roadmap. This roadmapserves as a guide in pharmacological intervention in the intermediatefilament or basal cell transcriptional machinery. It also points totransformed il+ cells as a source for pulmonary epithelial cells.

Functional Characteristics of R2R¹ and R2R²

The VEGF-A (mouse isoform VEGF¹⁶⁴=isoform VEGF¹⁶⁵) dependent expressionof the intermediate filament group of genes has been confirmed in themouse embryo. Expression of these intermediate filament genes leads todifferentiation of pulmonary epithelial cells and development of thebasal cell program in pulmonary mesenchymal cells.

Furthermore, VEGF-A (mouse isoform VEGF¹⁶⁴=isoform VEGF¹⁶⁵) dependentexpression of intermediate filament genes has also been confirmed inadult human primary epithelial cells. Stimulation of these cells byVEGF-A isoform VEGF¹⁶⁵ leads to upregulation of intermediate filamentgene expression. Intermediate filament gene expression in these cells isdownregulated by isoform VEGF¹⁶⁵ specific siRNA's. In summary, theexpression of intermediate filament genes serves as a paradigm of airwaydifferentiation and regeneration. R2R¹ and R2R² play a specific role inthis expression program.

R2R¹

The expression of R2R¹ is VEGF-A (mouse isoform VEGF¹⁶⁴=human isoformVEGF¹⁶⁵) dependent in the mouse. Mesenchymal cells of the lung (GS-IB4positive staining cells) acquire a basal epithelial cell gene expressionprogram. The expression of R2R¹ is essential for mesenchymal toepithelial transition (MET).

The role of R2R¹ in MET has now been confirmed in embryonic tissue otherthan the lung: completion of the ventricular septum of the heart isaccomplished by MET. In the mouse, R2R¹ is highly expressed in thedeveloping ventricular septum and is VEGF-A (mouse isoform VEGF¹⁶⁴=humanisoform VEGF¹⁶⁵) dependent. Contrary, R2R¹ expression is not found inthe developing right ventricular outflow tract: development of thisstructure is known to be MET independent (see FIG. 12)

R2R¹ is a candidate gene in mouse and human for mesenchymal toepithelial transition: the expression of this gene transduces the VEGF-A(mouse isoform VEGF¹⁶⁴=human isoform VEGF¹⁶⁵) effect on MET. The reverseprocess of MET is EMT (epithelial to mesenchymal transition). EMT is anessential part of cancer progression and metastasis. R2R¹ may thereforeplay an important role in cancer biology and therapy.

Furthermore, in silico analysis revealed that the protein product ofR2R¹ likely interacts with the ribosome. Aside from the scientificimpact, the interaction of the R2R¹ protein structure with the ribosomehas implications for drug development in the field of cancer biology andMET.

R2R²

R2R² has been found to be very highly expressed in adult human primarylung epithelial cells. In vitro, the expression of R2R¹ has been foundto be upregulated over time and to be VEGF-A (isoform VEGF¹⁶⁵)dependent. The R2R² protein product is important for normaldifferentiation and maintenance of the adult pulmonary epithelium (seeFIG. 13).

1-12. (canceled)
 13. An antibody or antigen binding fragment thereofcapable of binding to an R2R^(1/2) protein or a fragment thereof. 14.The antibody of claim 13, wherein the R2R^(1/2) protein is encoded by asequence selected from the group consisting of those designated SEQ IDNOS: 3, 6, 9 and 12, or a sequence at least 60% identical thereto.
 15. Amethod of treating one or more diseases and/or conditions selected fromthe group consisting of: (a) cardiac diseases and/or conditions; (b)pulmonary diseases and/or conditions; and (c) cancer; said methodcomprising administering a therapeutically effective amount of: (i) apolynucleotide comprising an R2R^(1/2) gene, wherein the R2R^(1/2) geneis encoded by a sequence selected from the group consisting of thosedesignated SEQ ID NOS: 1, 2, 4, 5, 7, 8, 10 and 11, or a sequence atleast 60% identical thereto; (ii) a polypeptide comprising an R2R^(1/2)protein, wherein the R2R^(1/2) protein is encoded by a sequence selectedfrom the group consisting of those designated SEQ ID NOS: 3, 6, 9 and 12or a sequence at least 60% identical thereto; (iii) an antisenseoligonucleotide capable of modulating the expression of an R2R^(1/2)gene; (iv) the antibody of claim 13; or (v) the antibody of claim 13,wherein the R2R^(1/2) protein is encoded by a sequence selected from thegroup consisting of those designated SEQ ID NOS: 3, 6, 9 and 12, or asequence at least 60% identical thereto; to a subject in need thereof.16. A method of diagnosing one or more diseases and/or conditionsselected from the group consisting of: (a) a cardiac disease and/orcondition; (d) a pulmonary disease and/or condition; (c) cancer; and (d)a susceptibility or predisposition to any of (a) to (c); said methodcomprising the step of detecting a level of R2R¹ and/or R2R² geneexpression in a sample provided by a subject to be tested, whereindetection of an aberrant level of the R2R¹ and/or R2R² gene in saidsample, is indicative of one or more of the diseases and/or conditionsprovided as (a)-(c) above and/or a susceptibility or predispositionthereto.
 17. The method of claim 16, wherein the method comprises theuse of an oligonucleotide probe and/or primer designed to hybridiseunder stringent conditions to all or part of a sequence selected fromthe group consisting of SEQ ID NOS: 1; 2; 4; 5; 7; 8; 10 and
 11. 18. Amethod of identifying or obtaining agents which modulate the expressionof an R2R¹ and/or R2R² genes, said method comprising the steps ofcontacting the R2R¹ and/or R2R² genes with a test agent and detectingany modulation of R2R^(1/2) gene expression.
 19. A pharmaceuticalcomposition comprising (i) an R2R^(1/2) gene provided by SEQ ID NOS: 1,2, 4, 5, 7, 8, 10 or 11, or a sequence at least 60% identical thereto,in association with a pharmaceutically acceptable excipient, carrier ordiluent; or (ii) an R2R^(1/2) protein provided by SEQ ID NOS: 3, 6, 9and 12, or a sequence at least 60% identical thereto, in associationwith a pharmaceutically acceptable excipient, carrier or diluent.
 20. Ananimal model for the study of a cardiac and/or pulmonary disease orcondition, or cancer, wherein the animal model has been generated bymodulation or disruption of an R2R^(1/2) gene.